Human Prt1-like subunit protein (hPrt1) polynucleotides

ABSTRACT

The present invention relates to novel human Prt1 (hPrt1) and eIF4G-like (p97) proteins which are involved in eukaryotic transcription. In particular, isolated nucleic acid molecules are provided encoding the human hPrt1 and p97 proteins. hPrt1 and p97 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of hPrt1 and p97 activity. Also provided are therapeutic methods for treating disease states associated with the hPrt1 and p97 proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.08/990,140, filed Dec. 12, 1997, now U.S. Pat. No. 6,093,795, which isherein incorporated by reference; said Ser. No. 08/990,140 claimspriority to U.S. Provisional Application No. 60/033,151, filed Dec. 13,1996, which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel proteins involved in theinitiation of eukaryotic transcription. More specifically, isolatednucleic acid molecules are provided encoding a human Prt1-like subunitprotein (hPrt1) and a human eIF4G-like protein (p97). Also provided arehPrt1 and p97 polypeptides, as are vectors, host cells and recombinantmethods for producing the same. The invention further relates toscreening methods for identifying agonists and antagonists of hPrt1 andp97 activity.

2. Related Art

Eukaryotic protein synthesis requires the participation of translationinitiation factors, which assist in the binding of the mRNA to the 40Sribosomal subunit (reviewed in Merrick & Hershey, in TranslationalControl, Hershey et al., eds., Cold Spring Harbour Laboratory Press,(1996), pp. 31-69 and Pain, Eur. J. Biochem 236:747-771 (1996)).Ribosome binding is facilitated by the cap structure (m⁷GpppN, where Nis any nucleotide) that is present at the 5′ end of all cellular mRNAs(except organellar). Biochemical fractionation studies elucidated thegeneral pathway for translation initiation and led to thecharacterization of several translation initiation factors (reviewed inMerrick & Hershey supra). It is believed that the mRNA cap structure isinitially bound by eukaryotic initiation factor (eIF) 4F, which, inconjunction with eIF4B, melts RNA secondary structure in the 5′untranslated region (UTR) of the mRNA to promote ribosome binding. eIF4Fis a more efficient RNA helicase than free eIF4A (Rozen et al., Mol.Cell. Biol. 10:1134-1144 (1990)), consistent with the idea that eIF4Arecycles through the eIF4F protein complex to function in unwinding(Pause et al., Nature 371:762-767 (1994)). The 40S ribosomal subunit. ina complex with eIF3, eIF1A and eIF2-GTP-tRNAimet, binds at or near thecap structure and scans vectorially the 5′ UTR in search of theinitiator AUG codon (reviewed in Merrick & Hershey, supra).

eIF3 is the largest translation initiation factor, with at least 8different polypeptide subunits and a total mass of approximately 550 to700 kDa (Schreier, et al., J. Mol. Biol. 116:727-753 (1977); Benne &Hershey, Proc. Natl. Acad. Sci. USA 73:3005-3009 (1976); Behlke et al.,Eur. J. Biochem. 157:523-530 (1986)). In mammals, the apparent molecularmasses of the eIF3 subunits are 35, 36, 40, 44, 47, 66, 115 and 170 kDa(Behlke, supra; Meyer, et al., Biochemistry 21:4206-4212 (1982); Milburnet al., Arch. Biochem. Biophys 276:6-11 (1990)). eIF3 is a moderatelyabundant translation initiation factor, with 0.5 to 1 molecule perribosome in HeLa cells and rabbit reticulocyte lysates (Meyer, supra;Mengod & Trachsel, Biochem. Acta 825:169-174 (1985)). This proteincomplex assumes several functions during translation initiation(reviewed in Hannig, BioEssays 17:915-919 (1995)). eIF3 binds to the 40Sribosomal subunit and prevents joining with the 60S subunit. Itinteracts with the ternary complex and stabilizes the binding of thelatter to the 40S ribosomal subunit (Trachsel et al., J. Mol. Biol.116:755-767 (1977); Gupta et al., (1990); Goumans et al., Biochem.Biophys. Acta 608:39-46 (1980); Peterson et al., J. Biol. Chem.254:2509-2510 (1979)). eIF3 crosslinks to mRNA and 18S mRNA (Nygard &Westermann, Nucl. Acids Res. 10:1327-1334 (1982); Westermann & Nygard,Nucl. Acids Res. 12:8887-8897 (1984)), an activity mainly attributed tothe 66 kDa subunit (or 62 kDa in yeast; Garcia-Barrio, et al., GenesDev. 9:1781-1796 (1995); Naranda, et al., J. Biol. Chem. 269:32286-32292(1994)). eIF3 co-purifies with eIF4F and eIF4B, two initiation factorsinvolved in the mRNA binding step (Schreier et al., J. Mol. Biol.116:727-753 (1977)). A direct interaction between the 220 kDa subunit ofeIF4F and eIF3 has been demonstrated (Lamphear et al., J. Biol. Chem.270:21975-21983 (1995)) and a role for eIF3 serving as a bridge betweenthe 40S ribosomal subunit and eIF4F-bound mRNA has been postulated(Lamphear, supra).

The complex structure of eIF3 and its pleiotropic roles in translationinitiation have rendered the study of this factor difficult. The proteinsequence for only three of the yeast subunits (SUI1/p16, p62 andPRT1/p90) have been published (Garcia-Barrio et al., Genes Dev.9:1781-1796 (1995); Naranda, supra; Hanic-Joyce et al., J. Biol. Chem.262:2845-2851 (1987)). However, several other mammalian and yeastsubunits have been recently cloned. The yeast protein p90, also known asPrt1, is the most well characterized of those identified to date. Prt1is an integral subunit of eIF3 (Naranda, supra; Danaie et al., J. Biol.Chem. 270:4288-4292 (1995)). A conditional lethal mutation in the PRT1gene reduces the binding of the ternary to the 40S ribosomal subunit(Feinberg et al., J. Biol. Chem. 257:10846-10851 (1982)). Othermutations which confer temperature sensitivity are located in thecentral and carboxy-terminal portion of Prt1. An N-terminal deletionwhich removes the Prt1 putative RNA Recognition Motif (RRM; for reviewssee Birney, et al., Nucl. Acids Res. 21:5803-5816 (1993); Burd &Dreyfuss, Science 265:615-621 (1994b); Nagai et al., Trends Biochem.Sci. 20:235-240 (1995)), acts a trans-dominant negative inhibitor (Evanset al., Mol. Cell. Biol. 15:4525-4535 (1995)).

Proteins that specifically inhibit cap-dependent translation have beendescribed (Pause, supra; Lin et al., Science 266:653-656 (1994)):4E-binding protein-1 and -2 (4E-BP1 and 4E-BP2) bind to eIF4E andprevent their association with eIF4G, because 4E-BPs and eIF4G share acommon site for eIF4E binding (Haghighat et al., EMBO J. 14:5701-5709(1995); Mader et al., Mol. Cell. Biol. 15:4990-4997 (1995)). Upontreatment of cells with insulin and growth factors, 4E-BPs becomephosphorylated. This leads to dissociation of the 4E-BPs from eIF4E andformation of the eIF4F complex, which results in stimulation oftranslation (Pause, supra; Lin, supra; Beretta, et al., EMBO J.15:658-664 (1996)).

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising polynucleotides encoding the hPrt1 and p97 polypeptideshaving the amino acid sequences shown in FIGS. 1A-1D (SEQ ID NO:2) andFIGS. 2A-2E (SEQ ID NO:4) or the amino acid sequences encoded by thecDNA clones deposited as ATCC Deposit Number 97766 on Oct. 18, 1996 andATCC Deposit Number 97767 on Oct. 18, 1996.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofhPrt1 and p97 polypeptides or peptides by recombinant techniques.

The invention further provides isolated hPrt1 and p97 polypeptideshaving amino acid sequences encoded by polynucleotides described herein.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a cellular response inducedby hPrt1 and/or p97 polypeptides, which involves contacting cells whichexpress hPrt1 and/or p97 polypeptides with the candidate compound,assaying a cellular response, and comparing the cellular response to astandard cellular response, the standard being assayed when contact ismade in absence of the candidate compound; whereby, an increasedcellular response over the standard indicates that the compound is anagonist and a decreased cellular response over the standard indicatesthat the compound is an antagonist.

Additional aspects of the invention relate to methods for treating anindividual in need of either an increased or decreased level of hPrt1and/or p97 activity in the body comprising administering to such anindividual a composition comprising a therapeutically effective amountof either an isolated hPrt1 and/or p97 polypeptides of the invention (oran agonist thereof) or an hPrt1 and/or p97 antagonist.

The present invention also provides components for use in in vitrotranslation systems. Two individual components of such translationsystems, hPrt1 and p97, are provided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the nucleotide (SEQ ID NO:1) and deduced amino acid(SEQ ID NO:2) sequence of the hPrt1 polypeptide. The protein has amolecular weight of about 116 kDa, as shown in Example 1. The standardone-letter abbreviations for amino acids are used.

FIGS. 2A-2E show the nucleotide (SEQ ID NO:3) and deduced amino acid(SEQ ID NO:4) sequence of the p97 polypeptide. The protein has amolecular weight of about 97 kDa, as shown in Example 2. Abbreviationsare as in FIGS. 1A-1D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding an hPrt1 polypeptide having theamino acid sequence shown in FIGS. 1A-1D (SEQ ID NO:2). The hPrt1protein of the present invention shares sequence homology with the Prt1protein of Saccharomyces cerevisiae. The nucleotide sequence shown inFIGS. 1A-1D (SEQ ID NO:1) was obtained by sequencing a cDNA clone, whichwas deposited on Oct. 18, 1996 at the American Type Culture Collection,10801 University Blvd., Manassas Va. 20110-2209, USA and given accessionnumber 97766. The deposited clone is contained in the pBluescript SK(−)plasmid (Stratagene, LaJolla, Calif.).

In addition, the present invention also provides isolated nucleic acidmolecules comprising a polynucleotide encoding an p97 polypeptide havingthe amino acid sequence shown in FIGS. 2A-2E (SEQ ID NO:4). The p97protein of the present invention shares sequence homology with the humaneIF4G protein. The nucleotide sequence shown in FIGS. 2A-2E (SEQ IDNO:3) was obtained by sequencing a cDNA clone, which was deposited onOct. 18, 1996 at the American Type Culture Collection, 10801 UniversityBlvd., Manassas Va. 20110-2209, USA and given accession number 97767.The deposited clone is contained in the pcDNAIII plasmid (Invitrogen,Inc.).

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-1D (SEQ ID NO:1), nucleic acid molecules of the presentinvention encoding the hPrt1 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. While the hPRT1 gene was found to be presentin cDNA libraries produced from RNA from multiple tissues, the nucleicacid molecule described in FIGS. 1A-1D (SEQ ID NO:1) was isolated from acDNA library derived from human bone marrow cells. The determinednucleotide sequence of the hPrt1 cDNA of FIGS. 1A-1D (SEQ ID NO:1)contains an open reading frame encoding a protein of 873 amino acidresidues, with an initiation codon at positions 97-99 of the nucleotidesequence in FIGS. 1A-1D (SEQ ID NO:1) and a molecular weight of about116 kDa, as shown in Example 1. The hPrt1 protein shown in FIGS. 1A-1D(SEQ ID NO:2) is about 31% identical and about 50% similar to the Prt1protein of Saccharomyces cerevisiae (GenBank Accession No. J02674).

In addition, using the information provided herein, such as thenucleotide sequence in FIGS. 2A-2E (SEQ ID NO:3), a nucleic acidmolecule of the present invention encoding a p97 polypeptide may also beobtained using standard cloning and screening procedures. While the p97gene was identified in cDNA libraries produced from RNA from severaltissues, the nucleic acid molecule described in FIGS. 2A-2E (SEQ IDNO:3) was isolated from a cDNA library derived from human fetal heart.The determined nucleotide sequence of the p97 cDNA of FIGS. 2A-2E (SEQID NO:3) contains an open reading frame encoding a protein of 907 aminoacid residues, with an initiation codon at positions 307-309 of thenucleotide sequence in FIGS. 2A-2E (SEQ ID NO:3) and a molecular weightof about 97 kDa, as shown in Example 2. The p97 protein shown in FIGS.2A-2E (SEQ ID NO:4) is about 28% identical and about 36% similar toapproximately the C-terminal two thirds of eIF4G (GenBank Accession No.

D12686). The N-terminal third of eIF4G bears no similarity to the p97protein of the present invention.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above the actual hPrt1 polypeptide encodedby the deposited cDNA comprises about 873 amino acids, but may beanywhere in the range of about 850 to about 896 amino acids. Similarly,the actual p97 polypeptide encoded by the deposited cDNA comprises about907 amino acids, but may be anywhere in the range of about 882 to about932 amino acids.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. Single-stranded DNA or RNAmay be the coding strand, also known as the sense strand, or it may bethe non-coding strand, also referred to as the anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.

Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules further includes such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising open reading frames (ORF) with initiation codons atpositions 97-99 of the nucleotide sequence shown in FIGS. 1A-1D (SEQ IDNO:1) for hPrt1 and positions 307-309 of the nucleotide sequence shownin FIGS. 2A-2E (SEQ ID NO:3) for p97; and DNA molecules which comprise asequence substantially different from those described above but which,due to the degeneracy of the genetic code, still encode either the hPrt1or p97 proteins.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the hPrt1 and p97 polypeptides having amino acidsequences encoded by the cDNA clones contained in the plasmids depositedas ATCC Deposit No. 97766 on Oct. 18, 1996 and ATCC Deposit No. 97767 onOct. 18, 1996, respectively. The invention further provides isolatednucleic acid molecules having the nucleotide sequences shown in FIGS.1A-1D (SEQ ID NO:1), FIGS. 2A-2E (SEQ ID NO:3), the nucleotide sequenceof the hPrt1 and p97 cDNA contained in the above-described depositedclones, or a nucleic acid molecule having a sequence complementary toany one of the above sequences. In a further embodiment, isolatednucleic acid molecules are provided encoding the full-length hPrt1 andp97 polypeptides lacking the N-terminal amino acid residue.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequences of the depositedcDNAs or the nucleotide sequences shown in FIGS. 1A-1D (SEQ ID NO:1) orFIGS. 2A-2E (SEQ ID NO:3) is intended fragments at least about 15 nt,and more preferably at least about 20 nt, still more preferably at leastabout 30 nt, and even more preferably, at least about 40 nt in lengthwhich are useful as diagnostic probes and primers as discussed herein.Of course larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750,1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350,2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950,3000, or 3010 nt in length of the sequence shown in SEQ ID NO:1 are alsouseful according to the present invention as are fragments correspondingto most, if not all, of the nucleotide sequence of the cDNA clonecontained in the plasmid deposited as ATCC Deposit No. 97766 or as shownin SEQ ID NO:1. Similarly, larger DNA fragments 50, 100, 150, 200 ,250,300, 350, 400, 450, 500, 550. 600, 650, 700, 750, 800, 850, 900, 100,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200,2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800,2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400,3450, 3500, 3550, 3600, 3650, 3700, 3750, or 3790 nt in length of thesequence shown in SEQ ID NO:3 are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the cDNA clone contained in the plasmid depositedas ATCC Deposit No. 97767 or as shown in SEQ ID NO:3. By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from the nucleotide sequences of thedeposited cDNAs or the nucleotide sequences as shown in FIGS. 1A-1D (SEQID NO:1) or FIGS. 2A-2E (SEQ ID NO:3). Since the genes have beendeposited and the nucleotide sequences shown in FIGS. 1A-1D (SEQ IDNO:1) and FIGS. 2A-2E (SEQ ID NO:3) are provided, generating such DNAfragments would be routine to the skilled artisan.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the hPrt1protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 1 to about 188 in FIGS. 1A-1D(SEQ ID NO:2); a polypeptide comprising amino acid residues from about193 to about 235 in FIGS. 1A-1D (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 248 to about 262 in FIGS. 1A-1D (SEQ IDNO:2); a polypeptide comprising amino acid residues from about 270 toabout 350 in FIGS. 1A-1D (SEQ ID NO:2); a polypeptide comprising aminoacid residues from about 361 to about 449 in FIGS. 1A-1D (SEQ ID NO:2);

a polypeptide comprising amino acid residues from about 458 to about 620in FIGS. 1A-1D (SEQ ID NO:2); and a polypeptide comprising amino acidresidues from about 639 to about 846 in FIGS. 1A-1D (SEQ ID NO:2). Theinventors have determined that the above polypeptide fragments areantigenic regions of the hPrt1 protein. Methods for determinig othersuch epitope-bearing portions of the hPrt1 protein are described indetail below.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding epitope-bearing portions of the p97protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 1 to about 98 in FIGS. 2A-2E(SEQ ID NO:4); a polypeptide comprising amino acid residues from about121 to about 207 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprisingamino acid residues from about 232 to about 278 in FIGS. 2A-2E (SEQ IDNO:4); a polypeptide comprising amino acid residues from about 287 toabout 338 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprising aminoacid residues from about 347 to about 578 in FIGS. 2A-2E (SEQ ID NO:4);a polypeptide comprising amino acid residues from about 593 to about 639in FIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprising amino acidresidues from about 681 to about 770 in FIGS. 2A-2E (SEQ ID NO:4); apolypeptide comprising amino acid residues from about 782 to about 810in FIGS. 2A-2E (SEQ ID NO:4); and a polypeptide comprising amino acidresidues from about 873 to about 905 in FIGS. 2A-2E (SEQ ID NO:4). Theinventors have determined that the above polypeptide fragments areantigenic regions of the p97 protein. Methods for determining other suchepitope-bearing portions of the p97 protein are also described in detailbelow.

In another aspect, the invention provides isolated nucleic acidmolecules comprising polynucleotides which hybridize under stringenthybridization conditions to a portion of a polynucleotide in nucleicacid molecules of the invention described above, for instances, the cDNAclones contained in ATCC Deposits Nos. 97766 and 97767. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (750 m NaCl, 75 mM trisodiumCitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

By polynucleotides which hybridize to a “portion” of a polynucleotide isintended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNAs or the nucleotide sequences as shown in FIGS. 1A-1D (SEQ ID NO:1)and FIGS. 2A-2E (SEQ ID NO:3)). Of course, a polynucleotide whichhybridizes only to a poly A sequence (such as the 3′ terminal poly(A)tract of the hPrt1 and p97 cDNAs, shown in FIGS. 1A-1D (SEQ ID NO:1) andFIGS. 2A-2E (SEQ ID NO:3)), or to a complementary stretch of T (or U)resides, would not be included in polynucleotides of the invention usedto hybridize to a portion of a nucleic acid of the invention, since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

Nucleic acid molecules of the present invention which encode the hPrt1and p97 polypeptides may include, but are not limited to those encodingthe amino acid sequences of the mature polypeptides, by themselves; thecoding sequences for the mature polypeptides and additional sequences,such as those encoding amino acid leaders or secretory sequences, suchas a pre-, or pro- or prepro-protein sequences; the coding sequences ofthe mature polypeptides, with or without the aforementioned additionalcoding sequences, together with additional, non-coding sequences,including for example, but not limited to introns and non-coding 5′ and3′ sequences, such as the transcribed, non-translated sequences thatplay a role in transcription, mRNA processing, including splicing andpolyadenylation signals, for example—ribosome binding and stability ofmRNA; an additional coding sequence which codes for additional aminoacids, such as those which provide additional functionalities. Thus, thesequences encoding the polypeptides of the present invention may befused to a marker sequence, such as a sequence encoding a peptide whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (Qiagen, Inc.), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37: 767 (1984). As discussed below, other such fusion proteinsinclude the hPrt1 or p97 fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the hPrt1 and p97 proteins. Variants may occur naturally,such as a natural allelic variant. By an “allelic variant” is intendedone of several alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the full-lengthhPrt1 or p97 polypeptide having the complete amino acid sequence inFIGS. 1A-1D (SEQ ID NO:2) (amino acid residues from about 1 to about873) and FIGS. 2A-2E (SEQ ID NO:4) (amino acid residues from about 1 toabout 907); (b) a nucleotide sequence encoding the full-length hPrt1 orp97 polypeptide having the complete amino acid sequence in FIGS. 1A-1D(SEQ ID NO:2) (amino acid residues from about 2 to about 873) and FIGS.2A-2E (SEQ ID NO:4) (amino acid residues from about 2 to about 907) butlacking the N-terminal amino acid residue; (c) a nucleotide sequenceencoding the hPrt1 or p97 polypeptide having the amino acid sequenceencoded by the cDNA clones contained in ATCC Deposit Nos. 97766 and97767, respectively; or (d) a nucleotide sequence complementary to anyof the nucleotide sequences in (a), (b) or (c).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding either anhPrt1 or p97 polypeptide is intended that the nucleotide sequence of apolynucleotide which is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequences encoding thehPrt1 or p97 polypeptides. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequences shown in FIGS. 1A-1D (SEQ ID NO:1) and FIGS. 2A-2E(SEQ ID NO:3) or to the nucleotide sequences of the deposited cDNAclones can be determined conventionally using known computer programssuch as the Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981)), to find the best segment of homology between twosequences. When using Bestfit or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set, of course, such that the percentage of identity iscalculated over the full length of the reference nucleotide sequence andthat gaps in homology of up to 5% of the total number of nucleotides inthe reference sequence are allowed.

The present application is directed to nucleic acid molecules at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shownin FIGS. 1A-1D (SEQ ID NO:1), FIGS. 2A-2E (SEQ ID NO:3), or to thenucleic acid sequence of the deposited cDNAs, irrespective of whetherthey encode a polypeptide having hPrt1 or p97 activity. This is becauseeven where a particular nucleic acid molecule does not encode apolypeptide having such activity, one of skill in the art would stillknow how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer.Preferred, however, are nucleic acid molecules having sequences at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shownin FIGS. 1A-1D (SEQ ID NO:1), FIGS. 2A-2E (SEQ ID NO:3), or to thenucleic acid sequences of the deposited cDNAs which do, in fact, encodea polypeptide having hPrt1 or p97 protein activity. By “a polypeptidehaving hPrt1 or p97 activity” is intended polypeptides exhibitingactivity similar, but not necessarily identical, to an activity ofeither the hPrt1 or p97 protein of the invention, as measured in aparticular biological assay. For instance, p97 protein activity can bemeasured using the ability of a p97 homolog to either suppresstranslation or bind to eIF4A or eIF3, as described in the Examplesbelow. hPrt1 protein activity can be measured, for example, using theability of an hPrt1 homolog to interact with proteins in the eIF3complex, also as described in the Examples below.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or99% identical to the nucleic acid sequences of the deposited cDNAs orthe nucleic acid sequences shown in 1F-1D. (SEQ ID NO:1) or FIGS. 2A-2E(SEQ ID NO:3) will encode a polypeptide “having hPrt1 or p97 proteinactivity.” In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, this will be clear to theskilled artisan even without performing the above described comparisonassay. It will be further recognized in the art that, for such nucleicacid molecules that are not degenerate variants, a reasonable numberwill also encode polypeptides having hPrt1 or p97 protein activity.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that thereare two main approaches for studying the tolerance of an amino acidsequence to change. The first method relies on the process of evolution,in which mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality. These studies haverevealed that proteins are surprisingly tolerant of amino acidsubstitutions. The authors further indicate which amino acid changes arelikely to be permissive at a certain position of the protein. Numerousphenotypically silent substitutions are described in Bowie, J. U. etal., supra, and the references cited therein.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of hPrt1 andp97 polypeptides or fragments thereof by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. The DNA insert should be operativelylinked to an appropriate promoter, such as the phage lambda PL promoter,the E. coli lac, trp and tac promoters, the SV40 early and latepromoters and promoters of retroviral LTRs. to name a few. Othersuitable promoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the mature transcripts expressed bythe constructs will preferably include a translation initiating at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL-5 receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995)and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270,No. 16:9459-9471 (1995).

The hPrt1 and p97 proteins can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost. including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated.

hPrt1 and p97 Polypeptides and Fragments

The invention further provides isolated hPrt1 and p97 polypeptideshaving the amino acid sequences encoded by the deposited cDNAs, or theamino acid sequences shown in FIGS. 1A-1D (SEQ ID NO:2) and FIGS. 2A-2E(SEQ ID NO:4), or a peptide or polypeptide comprising a portion of theabove polypeptides.

It will be recognized in the art that some amino acid sequences of thehPrt1 and p97 polypeptides can be varied without significant effect onthe structure or function of the proteins. Thus, the invention furtherincludes variations of the hPrt1 and p97 polypeptides which showsubstantial hPrt1 and p97 polypeptide activities or which includeregions of hPrt1 and p97 proteins such as the portions discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions. As indicated above, guidance concerning which aminoacid changes are likely to be phenotypically silent can be found inBowie, J. U., et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

Thus, the fragment, derivative or analog of the polypeptides of FIGS.1A-1D (SEQ ID NO:2), FIGS. 2A-2E (SEQ ID NO:4), or those encoded by thedeposited cDNAs, may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the polypeptide is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the polypeptide.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the hPrt1 and p97 proteins. Theprevention of aggregation is highly desirable. Aggregation of proteinsnot only results in a loss of activity but can also be problematic whenpreparing pharmaceutical formulations, because they can be immunogenic.(Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al.,Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic DrugCarrier Systems 10:307-377 (1993)).

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table I).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above andbelow. Generally speaking, the number of substitutions for any givenhPrt1 or p97 polypeptide, or mutant thereof, will not be more than 50,40, 30, 20, 10, 5, or 3, depending on the objective.

Amino acids in the hPrt1 and p97 proteins of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such the ability to bind to cellular transcription factors orRNA.

The polypeptides of the present invention are preferably provided in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced andcontained within a recombinant host cell would be considered “isolated”for purposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host. For example, recombinantlyproduced versions of the hPrt1 and p97 polypeptides can be substantiallypurified by the one-step method described in Smith and Johnson, Gene67:31-40 (1988).

The polypeptides of the present invention include the polypeptidesencoded by the deposited cDNAs; a polypeptide comprising amino acidsfrom about 1 to about 873 in SEQ ID NO:2; a polypeptide comprising aminoacids from about 1 to about 907 in SEQ ID NO:4; a polypeptide comprisingamino acids from about 2 to about 873 in SEQ ID NO:2; a polypeptidecomprising amino acids from about 2 to about 907 in SEQ ID NO:4; as wellas polypeptides which are at least 90% or 95% identical, more preferablyat least 96% identical, still more preferably at least 97%, 98% or 99%identical to those described above and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

The polypeptides of the present inventions include polypeptides at least90% or 95% identical, more preferably at least 96% identical, still morepreferably at least 97%, 98% or 99% identical to either the polypeptidesencoded by the deposited cDNAs or the polypeptides of FIGS. 1A-1D (SEQID NO:2) or FIGS. 2A-2E (SEQ ID NO:4), and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an hPrt1 or p97polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of either the hPrt1 or p97polypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acidsequence shown in FIGS. 1A-1D (SEQ ID NO:2), FIGS. 2A-2E (SEQ ID NO:4)or to the amino acid sequence encoded by one of the deposited cDNAclones can be determined conventionally using known computer programssuch the Bestfit program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

The polypeptides of the present invention are useful as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of the polypeptides of theinvention. The epitope of these polypeptide portions is an immunogenicor antigenic epitope of polypeptides described herein. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. On the other hand, aregion of a protein molecule to which an antibody can bind is defined asan “antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

It is well known in that art that relatively short synthetic peptidesthat mimic part of a protein sequence are routinely capable of elicitingan antiserum that reacts with the partially mimicked protein. See, forinstance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.,Science 219:660-666 (1983). Peptides capable of elicitingprotein-reactive sera are frequently represented in the primary sequenceof a protein, can be characterized by a set of simple chemical rules,and are confined neither to immunodominant regions of intact proteins(i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to polypeptides of the invention. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about at least about 15 to about30 amino acids contained within the amino acid sequence of polypeptidesof the invention.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate hPrt1-specific antibodies include: a polypeptidecomprising amino acid residues from about 1 to about 188 in FIGS. 1A-1D(SEQ ID NO:2); a polypeptide comprising amino acid residues from about193 to about 235 in FIGS. 1A-1D (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 248 to about 262 in FIGS. 1A-1D (SEQ IDNO:2); a polypeptide comprising amino acid residues from about 270 toabout 350 in FIGS. 1A-1D (SEQ ID NO:2); a polypeptide comprising aminoacid residues from about 361 to about 449 in FIGS. 1A-1D (SEQ ID NO:2);apolypeptide comprising amino acid residues from about 458 to about 620in FIGS. 1A-1D (SEQ ID NO:2); and a polypeptide comprising amino acidresidues from about 639 to about 846 in FIGS. 1A-1D (SEQ ID NO:2). Asindicated above, the inventors have determined that the abovepolypeptide fragments are antigenic regions of the hPrt1 protein.

In addition, non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate p97-specific antibodies include: apolypeptide comprising amino acid residues from about 1 to about 98 inFIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residuesfrom about 121 to about 207 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptidecomprising amino acid residues from about 232 to about 278 in FIGS.2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residues fromabout 287 to about 338 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptidecomprising amino acid residues from about 347 to about 578 in FIGS.2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residues fromabout 593 to about 639 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptidecomprising amino acid residues from about 681 to about 770 in FIGS.2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residues fromabout 782 to about 810 in FIGS. 2A-2E (SEQ ID NO:4); and a polypeptidecomprising amino acid residues from about 873 to about 905 in FIGS.2A-2E (SEQ ID NO:4). As indicated above, the inventors have determinedthat the above polypeptide fragments are antigenic regions of the p97protein.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. Houghten, R. A., Proc. Natl. Acad.Sci. USA 82:5131-5135 (1985). This “Simultaneous Multiple PeptideSynthesis (SMPS)” process is further described in U.S. Pat. No.4,631,211.

As one of skill in the art will appreciate, the hPrt1 and p97polypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.Fusion proteins that have a disulfide-linked dimeric structure due tothe IgG part can be more efficient in binding and neutralizing othermolecules than the monomeric protein or protein fragment alone(Fountoulakis et al., J. Biochem 270:3958-3964 (1995)).

hPrt1 and p97Polypeptides: Use for Screening for Agonists andAntagonists of hPrt1 and p97 Polypeptide Function

In one aspect, the present invention is directed to a method forenhancing an activity of an hPrt1 (e.g., modulation of apoptosis, theability to bind RNA or other known cellular ligands (such as p170 andeIF4G), participation in the process of translation) or p97 (e.g.,modulation of apoptosis, the ability to bind eIF4A and eIF3, suppressionof translation) polypeptide of the present invention, which involvesadministering to a cell which expresses the hPrt1 and/or p97 polypeptidean effective amount of an agonist capable of increasing an activity ofeither the hPrt1 or p97 protein. Preferably, the hPrt1 or p97polypeptide mediated activity is increased to treat a disease.

In a further aspect, the present invention is directed to a method forinhibiting an activity of an hPrt1 or p97 polypeptide of the presentinvention, which involves administering to a cell which expresses thehPrt1 and/or p97 polypeptide an effective amount of an antagonistcapable of decreasing an activity of either the hPrt1 or p97 protein.Preferably, hPrt1 or p97 polypeptide mediated activity is decreased toalso treat a disease.

By “agonist” is intended naturally occurring and synthetic compoundscapable of enhancing or potentiating an activity of an hPrt1 or p97polypeptide of the present invention. By “antagonist” is intendednaturally occurring and synthetic compounds capable of inhibiting anactivity of an hPrt1 or p97 polypeptide. Whether any candidate “agonist”or “antagonist” of the present invention can enhance or inhibit anactivity can be determined using art-known assays, including thosedescribed in more detail below.

Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular activity of an hPrt1 or p97polypeptide. The method involves contacting cells which express one orboth of the hPrt1 or p97 polypeptides with a candidate compound,assaying a cellular response, and comparing the cellular response to astandard cellular response, the standard being assayed when contact ismade with the polypeptide(s) in absence of the candidate compound,whereby an increased cellular response over the standard indicates thatthe candidate compound is an agonist of the polypeptide activity and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the activity. By “assaying acellular response” is intended qualitatively or quantitatively measuringa cellular response to a candidate compound and either an hPrt1 or p97polypeptide (e.g., modulation of apoptosis, the ability to bind RNA orother known cellular ligands, participation in the process oftranslation).

Potential antagonists include the hPrt1 RRM and fragments thereof, e.g.,hPrt1 polypeptide fragments that include the RNA binding domain. Suchforms of the protein, which may be naturally occurring or synthetic,antagonize hPrt1 polypeptide mediated activity by competing for bindingto RNA. Thus, such antagonists include fragments of the hPrt1 thatcontain the ligand binding domains of the polypeptides of the presentinvention.

Additional agonists according to the present invention include fragmentsof the p97 polypeptide capable of suppressing translation.

Proteins and other compounds which bind the hPrt1 or p97 polypeptidedomains are also candidate agonist and antagonist according to thepresent invention. Such binding compounds can be “captured” using theyeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989);Gyuris et al., Cell 75:791-803 (1993); Zervos et al., Cell 72:223-232(1993)). hPrt1 and p97 polypeptide antagonists also include smallmolecules which bind to and occupies active regions of the hPrt1 or p97polypeptide thereby making the polypeptide inaccessible to ligands whichbind thereto such that normal biological activity is prevented. Examplesof small molecules include but are not limited to nucleotide sequencesand small peptides or peptide-like molecules. Such molecules may beproduced and screened for activity by a variety of methods (e.g., Lightand Lerner, Bioorganic & Medicinal Chemistry 3 (7):955-967 (1995); Chenget al., Gene 171:1-8 (1996); Gates et al., J. Mol. Biol. 255:373-386(1996)).

In Vitro Translation Systems

The polypeptides of the present invention are also valuable for use inin vitro translation systems. The events leading to the initiation ofprotein synthesis in eukaryotic cells have been studied using bothreconstitution of translational systems using purified components and,more recently, genetic analyses. Hannig, BioEssays 17(11):915-919(1995). There remains, however, a current need for identifying moleculesinvolved in translation and the role each of those molecules play in theprocess of translation. The present invention provides two suchmolecules: hPrt1 and p97.

Several commercially available kits are currently on the market forperforming translation in vitro. See, e.g., Boehringer Mannheim,Indianapolis, Ind., Cat. Nos:1559 451, 1103 059, 1103 067; LifeTechnologies, Grand Island, N.Y., Cat. Nos:18127-019, 18128-017. Thesekits generally provide lysates derived from either whole animal or plantcells which are capable of producing protein from mRNA. These lysatesare generally used as part of a translation reaction mixture whichcontains, in addition to the lysate, mRNA and both labeled and unlabeledamino acids. Thus, while the process of translation can generally beperformed to produce a protein of interest, the mechanism by which thoseproteins are produced has not yet been fully elucidated.

The present invention provides individual components of these celllysates which are useful for studying the process of translation. Thep97 protein, for example, as a putative competitive inhibitor of eIF4Gwhich suppresses both cap dependent and independent translation, isuseful for identifying mechanisms by which gene expression is regulatedat the translational level. The p97 protein may also be useful foridentifying specific genes which are regulated at the translationallevel.

Similarly, the present invention also provides the hPrt1 protein whichis believed to be both a member of the eIF3 complex and a necessarycomponent of translation systems. In order for researchers to fullyreconstitute a translation system from individual proteins each of theproteins of that system must be identified and available in purifiedform. The present invention provides the hPrt1 protein as one of thosecomponents. Such reconstitution studies will be useful in elucidatingthe specific role of each component of the system. For example, theprocesses of both initiation of proteins synthesis and elongation of theresulting polypeptide chain can be studied by either altering the ratiosof the various components or leaving one or more component out of thereaction mixture.

In addition, the present invention provides fragments and homologs ofthe hPrt1 and p97 polypeptides, produced as described above, which actas either agonists or antagonists of the hPrt1 and p97 proteins. Suchfragments of the hPrt1 polypeptide may be useful, for example, forinhibiting translation by blocking the binding of native hPrt1 witheither other proteins to form the eIF3 complex or RNA. In addition, suchfragments of the p97 polypeptide may be useful, for example, forcompetitively inhibiting eIF4G.

Having generally described the invention. the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLE 1 Materials

Materials were obtained from the following sources: T7 DNA polymerasesequencing kit, Pharmacia LKB Biotechnologies. Protein A-Sepharose,Repligen. Heart muscle kinase, Sigma. Hybond-N+ nylon membrane,chemiluminescence system, Amersham. Poyvinylidene fluoride membrane,Millipore. γ³²P-ATP (6000 Ci/mmol), α³²P dCTP (3000 Ci/mmol),³⁵S-methionine (1000 Ci/mmol), DuPont-NEN. Oligonucleotides wereprepared at the Sheldon Biotechnology Centre, McGill University, Canada.

Isolation of hPrt1 cDNA clones

The expressed sequence tag used in this study (EST 112738 from HumanGenome Science (HGS) Inc.) was identified using established EST methodsdescribed previously (Adams, M. D., et al., Nature (London) 377:3-174(1995)), and this partial cDNA clone encoding the human homologue of theyeast Prt1 protein was used to obtain the full length cDNA clone. Fulllength cDNA clones for hPrt1 were isolated from a γgt11 human placentacDNA library. A 250 bp DNA was generated by the polymerase chainreaction (PCR) using the hPrt1 EST clone as template. The amplified DNAwas ³²P-labeled by random priming using a ³²p dCTP, random hexamers andthe Klenow fragment of DNA polymerase (Feinberg, A. P., & Vogelstein,B., Analytical Biochem. 137:266-267 (1984)), and used as a probe in cDNAscreening and Northern blot analysis. For cDNA screening, 5×10⁵ phagesdisplayed on duplicate sets of filters (Hybond-N+, Amersham) wereprehybridized in 5×SSPE (20×SSPE is 3.6 M NaCl, 0.2 M Na₃PO4, 0.02 MEDTA, pH 7.7), 5×Denhardt's solution (1×Denhardt's is 0.1% bovine serumalbumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), 0.5% SDS and 40 μg/mlheat-denatured salmon sperm DNA, for 4 hours at 65° C. Hybridization wasperformed in the same buffer containing the hPrt1 probe at 1×10⁶ cpm/mlfor 16 hours at 65° C. Filters were washed to a final stringency of0.1×SSPE/0.1% SDS at 65° C., and exposed to Kodak XAR films for 72 hourswith intensifying screens. Phages from positive clones were used toprepare plate lysates and DNA was purified, digested with SalI andligated into pBluescript that had been digested with SalI.Oligonucleotides used for sequencing were derived from eitherpBluescript or from the hPrt1 EST DNA sequence. The nucleotide sequencefor full length hPrt1-1 was obtained from both strands of independentoverlapping clones using the dideoxy chain termination method (Sanger,F., et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) and the T7polymerase sequencing kit (Pharmacia). Regions of compression werere-sequenced using 7-deaza dGTP.

Vectors, Proteins

The full length cDNA (clone 3-6) was excised from the γgt11 phage bySalI digestion and inserted into pBluescript KS in the T7 promoterorientation. The resulting vector is designated as KST7hPrt1-6.Constructs for truncated hPrt1 proteins were generated by PCR usingprimers in which an EcoRI site had been engineered. Cleavage of the PCRproduct with EcoRI and ligation into pAR90[59/69] (Blanar, M. A., &Rutter, W. J., Science 256:1014-1018 (1992)) or pGEX2T[128/129] (Blanar,M. A., & Rutter, W, supra) that had been digested with EcoRI preservesthe hPrt1 open reading frame and creates a GST-FLAG-HMK or FLAG-HMKfusion protein. For pAR90 N90146, the forward (5′) primer was 5′ACCGGAATTCAAAATGGACGCGGACGAGCCCTC 3′ (SEQ ID NO:5) and the reverse (3′)was primer 5′ AGCGGAATTCTTAAATCCCCCACTGCAG 3′ (SEQ ID NO:6). For pGEXN255, the hPrt1 open reading frame was first amplified by PCR andinserted into pGEX2T[128/129]. The resulting vector was linearized withHindIII, blunt ended with the Klenow fragment of E. coli DNA polymeraseand religated. Religation creates a stop codon 3 amino acids downstreamof hPrt1 residue 255. pGEX 146-255 was obtained by linearizing pGEXN90146 with HindIII, blunt-ending with Klenow and religating. Vectorswere transformed in either E. coli BL21 or BL21 pLysS. Bacteria weregrown in LB broth to an optical density of 0.5 and protein expressionwas induced with 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside) for 1 hat 37° C. Cells were pelleted and lysed in lysis buffer (PBS, 1 mM EDTA,1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride) by 6 sonication cycles.Debris was removed by centrifugation. GST fusion proteins were purifiedon glutathione-Sepharose (Pharmacia) as described previously (Methot,N., et al., Molecular and Cell Biology 14:2307-2316 (1994)). FLAG-HMKfusion proteins were affinity-purified over an anti-FLAG column (Kodak)according to the manufacturer's specifications. pACTAG-hPrt1 was made bylinearizing pACTAG-2 (Charest, A., et al., Biochem. J. 308:425-432(1995)) with NotI, and inserting the hPrt1 ORF that had been excisedfrom KST7hPrt1 cut with NotI. hPrt1 is expressed from this vector as afusion protein bearing three hemagglutinin (HA) tags at its aminoterminus.

In Vitro Transcription and Translation

KS17hPrt1-6 was digested with DraI, and the linearized plasmid was usedas template for in vitro transcription using T7 RNA polymerase (Promega)under conditions recommended by the supplier. Translation reactions wereperformed in nuclease-treated rabbit reticulocyte lysate (Promega) in afinal volume of 15 μl. Reaction mixtures contained 10 μl lysate, 10 μCi³⁵S-methionine (1000 Ci/Mmole), 15 U RNAsin (Promega), 20 μM amino acidmixture (minus methionine) and 100 ng RNA. The reactions were incubated60 minutes at 30° C. and stopped by the addition of 3 volumes of Laemmlibuffer. Translation products were analyzed by SDS-9% polyacrylamide gelelectrophoresis. Gels were fixed, treated in 16% salicylic acid, driedand processed for autoradiography.

Immunoprecipitations, Western Blots of hPrt1 Polypeptides

For HA-hPrt1 protein expression, HeLa cells that had been cultured inDubelco DMEM media supplemented with 10% fetal bovine serum (FBS) wereinfected for 1 hour with recombinant vaccinia virus vTF7-3 with the T7RNA polymerase cDNA inserted into its genome (Fuerst, T. R., et al.,Proc. Natl. Acad. Sci. USA 83:8122-8126 (1986)), and transfected with 5μg plasmid DNA using lipofectine (Gibco BRL, Gaithersburg, Md.) in DMEMwithout FBS. Cells were incubated 2 hours with the DNA-lipofectinemixture, and returned to DMEM-10% FBS for 12 hours before harvesting.The cells were lysed in 20 mM Tris-HCl pH 7.4, 75 mM KCl, 5 mM MgCl₂, 1mM DTT, 10 % glycerol, 1% Triton X-100, 1 mM PMSF, aprotinin (25 ng/ml)and pepstatin (1 ng/ml). Cellular debris and nuclei were removed bycentrifugation, and protein content was assayed by the Bradford method.Immunoprecipitations were performed on 200 μg of extract using a-HAantibody. Briefly, extracts were diluted to 500 μl in RIPA buffer (50 mMTris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% Na-deoxycholate)and incubated on ice for 30 minutes with 1 μg of antibody.Protein-A-sepharose was added and allowed to mix at 4° C. for 60minutes. The beads were washed 5 times in RIPA buffer before additionLaemmli buffer and boiling for 5 minutes. Immunoprecipitates were thenloaded on an SDS-10% polyacrylamide gel, blotted onto nitrocellulose andprobed with a goat anti-rabbit eIF3 antibody. Immunoreactive specieswere visualized using the Renaissance chemiluminescence system (ECL;Amersham). Affinity-purified antibodies against recombinant hPrt1 wereobtained as described in Harlow and Lane, ANTIBODIES: A LABORATORYMANUAL, Cold Spring Harbor (1988). E. coli extracts expressing hPrt1N90146 were fractionated by SDS-PAGE and transferred ontonitrocellulose. The bands containing N90146 were excised, blocked inBlotto (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.075% Tween-20, 0.5% milkpowder) and incubated with crude α-eIF3, and washed. Antibodies bound tothe membrane were eluted with 2 M glycine, 1 mM EGTA, pH 2.5, andneutralized by the addition of 1 M Tris-HCl, pH 8.8. To eliminatecontamination with p110 (hNip1), 50 μg of GST-p110 immobilizes onnitrocellulose were present during the incubation with crude α-eIF3antibodies. Western blotting was performed with antibodies at thefollowing dilutions: α-eIF3, 1:3000. α-p170, 1:10. For westerns blotsperformed with the monoclonal α-p170 antibody, horseradish-peroxidaseα-mouse 1 gM (Pierce) secondary antibodies were used; For α-eIF3, α-goatIgG-horseradish peroxidase; For α-hPrt1, α-goat IgG-alkalinephosphatase.

Northern Blot of hPrt1 mRNA

Total RNA from HeLa cells was isolated using Trizol (Life Technologies,Grand Island, N.Y.) and fractionated by electrophoresis in a 1%agarose/formaldehyde gel overnight at 40V. RNA was blotted to Hybond-N+filters overnight and UV cross-linked to the membrane using UV light.The membrane was prehybridized and hybridized under conditions identicalto the cDNA library screening, and exposed for 24 hours to a KodakBioMax film with intensifying screen.

Far Western Blots

Partially purified FLAG-HMK hPrt1 fusion proteins (1-3 μg) were ³²plabeled using heart muscle kinase as described (Blanar, M. A., & Rutter,W. J., supra). Proteins were resolved by SDS-polyacrylamide gelelectrophoresis and blotted on PVDF membranes (Millipore) ornitrocellulose. The membranes were blocked overnight with 5% milk in HBBbuffer (25 mM HEPES-KOH, pH 7.5, 25 mM NaCl, 5 mM MgCl₂, 1 mM DTF), andincubated 4 hours in hybridization buffer (20 mM HEPES-KOH pH 7.5, 75 mMKCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 0. 1% NP-40, 1% milk)containing the ³²P-labeled FLAG-HMK- or GST-FLAG-HMK-hPrt1 at 250,000cpm/ml and unlabeled purified GST at 1 μg/ml. The membranes were washed3 times with hybridization buffer and processed for autoradiography.

Results

Cloninig and Features of hPRT1

Expressed Sequence Tag (EST) #112738 from Human Genome Science (HGS)Inc. encodes a protein with homology to the yeast p90 eIF3 subunit,Prt1. The cDNA sequence, 2 kbp in length, contained a polyadenylationsignal and a short polyadenylate tail. An ATG codon was present at the5′ end of the clone. However, this ATG was not preceded by stop codons.It was therefore possible that EST #112738 contains an incomplete cDNA.A ³²P-labeled probe derived from the 5′ end of the EST sequence wasgenerated and used in a Northern analysis on HeLa cell RNA. A single RNAspecies migrating at 3.1 kb, hybridized with the probe. We concludedthat 1 kbp was missing from EST #112738. To obtain the full length cDNAsequence, a human placenta γgt11 cDNA library was screened with the sameprobe used in the Northern analysis. Forty positive were recovered andsome of the cDNAs extended further upstream of the 5′-most sequence ofEST #112738. One of these clones, 3-6, contained a 3 kbp insert with apredicted open reading frame of 873 amino acids, shown in FIGS. 1A-1D,(SEQ ID NO:2). An ATG codon, located at nucleotide positions 97-99downstream of the 5′ end, was preceded by an in-frame stop-codon(nucleotide positions 22 to 24, shown in FIGS. 1A-1D (SEQ ID NO:1).Thus, it is likely that clone 3-6 encodes the full length cDNA, and thefirst ATG constitutes the authentic initiation codon. An in frame CTGcodon 24 nt upstream of the first AUG is present and could potentiallyserve as the initiation site (nucleotide positions 73-75 shown in FIGS.1A-1D (SEQ ID NO:1)). We have named the protein encoded this cDNA hPrt1,for human-Prt1.

The cDNA sequence of hPrt1 is predicted to encode a protein containing acanonical RNA Recognition Motif (RRM) located between amino acids 185and 270 (FIGS. 1A-1D (SEQ ID NO:2)). The identification of the hPrt1 RRMis based on the consensus structural core sequences of RRMs (Birney, E.,et al., Nucl. Acids Res. 21:5803-5816 (1993)), which include thepresence of RNP-1 and RNP-2 sequence, and hydrophobic amino acids foundat specific positions within the RRM. A BLAST search (Altschul, S. F.,et al., J. Mol. Biol. 215:403-410 (1990)) with only the hPrt1 RRMrevealed that the hPrt1 RRM is most highly related to the fourth RRM ofthe poly(A) binding protein PABP. No other common protein motifs areevident. hPrt1 is acidic, with a predicted PI of 4.8. The middle portionof the protein is unusually rich in tryptophan residues (close to 5%tryptophan content over 400 amino acids). Amino acid sequence comparisonbetween human and Saccharomyces cerevisiae Prt1, reveal extensivesequence identity (31% identity, 50% homology) across the entire proteinexcept for the first 140 amino acids. The similarity between yeast andhuman Prt1 is more striking in the middle portion of the protein whichencompasses the RRM. Several but not all of the tryptophan areconserved, suggesting that they are functionally important. The aminoterminus of human Prt1 is not homologous to yeast Prt1, but insteadexhibits 25% identity to procollagen a chain precursor protein (data notshown). The significance of this is not clear. The hPrt1 protein alsocontains two protein kinase A, six protein kinase C and 17 casein kinaseII consensus phosphorylation sites.

hPrt1 is apart of eIF3

It is conceivable that hPrt1 is a subunit of eIF3. To prove this, animmunological characterization of hPrt1 and eIF3 was performed. First,we translated in vitro a synthetic RNA derived from the hPrt1 cDNA. Asingle polypeptide, migrating at 116 kDa on an SDS-9% polyacrylamidegel, was obtained. The translation product co-migrated with a 116 kDaprotein in a HeLa extract that cross-reacted with α-eIF3. Thus, the sizeof hPrt1 is similar to one of the eIF3 subunits. Next, we tested theability of a polyclonal α-eIF3 antibody to recognize hPrt1. To this end,we expressed hPrt1 fused to the hemagglutinin (HA) epitope-tag in HeLacells using a recombinant vaccinia virus expression system (Fuerst etal., supra). Extracts from infected cells were, blotted ontonitrocellulose and probed with a polyclonal α-eIF3 antibody. eIF3subunits (p 170 and p115) in extracts from cells transfected with theparental vector (pACTAG-2; Charest et al., Biochem. J. 308: 425-432(1995)) or pACTAG-hPrt1 were readily identifiable. A 125 kDa proteinthat cross-reacts with α-eIF3 was present in extracts from cellstransfected with pACTAG-hPrt1 but not in cells transfected with thevector alone. To confirm the identity of this protein as HA-hPrt1,immunoprecipitations using α-HA antibody were performed and the productsprobed with α-eIF3 antibody. The immunoprecipitated HA-hPrt1 co-migratedwith the 125 kDa polypeptide, and cross-reacted with the α-eIF3antibody. The slower mobility of HA-hPrt1 relative to hPrt1 is probablydue to the three HA epitopes present in the fusion protein.Immunoprecipitates from cells transfected with the parental vector orwith a vector encoding HA-La autoantigen failed to cross-react withα-eIF3. We conclude that hPrt1 is recognized by an antibody directedagainst eIF3. Finally we wished to determine whether antibodies directedagainst hPrt1 could recognize a 116 kDa polypeptide in purified humaneIF3. Attempts to generate antibodies against hPrt1 in rabbits failed.To circumvent this problem, affinity-purified hPrt1-specific antibodiesfrom α-eIF3 antisera were prepared from crude eIF3 antibodies, using abacterially expressed hPrt1 fragment. These antibodies recognized aprotein migrating at approximately 116 kDa in a highly purified humaneIF3 preparation and in HeLa extracts, and did not cross-react withhNip1 a 110 kDa protein also recently shown to be an eIF3 component (seethe discussion below). Together with the previous data, theseexperiments strongly suggest that hPrt1 is the 115 kDa subunit of eIF3.

hPrt1 Interacts Directly with the p170 Subunit of eIF3

To further substantiate the finding that hPrt1 is a subunit of humaneIF3, we examined the possibility that hPrt1 interacts directly with oneor more eIF3 subunits. To this end, hPrt1 was tagged with a FLAG peptidelinked to a heart muscle kinase site (FLAG-HMK) or fused to aglutathione-S-transferase-FLAG-HMK sequence (GST-FLAG-HMK). We opted touse fragments of hPrt1 rather than the full length protein due to thelow yield and extensive degradation of full length hPrt1 in E. coli. Theproteins were purified using a FLAG antibody or glutathione-sepharoseresin, and were ³²P-labeled with heart muscle kinase. The labeledproteins were then used to detect interacting proteins by the FarWestern assay with HeLa cytoplasmic extracts, rabbit reticulocyte lysateand different preparations of eIF3. Two of the probes, GST N255 (aminoacids 1-255 of hPrt1 shown in FIGS. 1A-1D (SEQ ID NO:2)) and N90146(amino acids 147-873 shown in FIGS. 1A-1D (SEQ ID NO:2)), interactedwith a 170 kDa protein in HeLa and rabbit reticulocyte lysate. The hPrt1probes reacted with a 140 kDa protein in eIF3 preparation 1 and 140 and170 kDa polypeptides in eIF3 preparation 2. A ³²P-labeled probeconsisting only of a GST-HMK fusion did not recognize any proteins (datanot shown).The 170 kDa protein in HeLa, rabbit reticulocytes and eIF3preparation 2 could the largest subunit of eIF3, p170. This protein issensitive to degradation, and the two eIF3 preparations used here differby the extent of p170 proteolysis. An immunoblot using a monoclonalantibody directed against p170 (Mengod, G., & Trachsel, H., Biochem.Acta 825:169-174 (1985)) revealed the extent of p170 degradation in theeIF3 preparations, and clearly shows that a 140 kDa degradation productof p170 is present in preparation 1, and to a lesser extent inpreparation 2. This experiment demonstrates that hPrt1 interactsdirectly with the p170 subunit of eIF3. No other eIF3 subunits wererecognized by the hPrt1 probes in this assay (data not shown).

The fact that both the N90146 and N255 fragments of hPrt1 reacted withp170 suggest that the site of protein-protein interaction is locatedbetween amino acids 147 and 255 (FIGS. 1A-1D (SEQ ID NO:2)). Thissegment of hPrt1, which encompasses most of the RRM, was assessed forits ability to interact with p170 independently of other sequences. Afragment containing amino acids 147 to 255 of hPrt1, used as a probe ina Far Western assay, indeed interacted with p170. To further delineatethe interaction site, a fragment consisting of amino acids 147-209(FIGS. 1A-1D (SEQ ID NO:2)) was tested, and failed to interact with p170(data not shown). This suggests that a portion of the RRM is crucial forthe association between hPrt1 and p170.

Discussion

The present invention provides a human cDNA that with homology to theyeast eIF3 subunit, Prt1. In vitro translation of hPrt1 RNA yielded apolypeptide of 116 kDa that co-migrated with one of the eIF3 subunits.Immunological characterization revealed that hPrt1 cross-reacts withα-eIF3 and that affinity-purified a-hPrt1 antibodies recognize apolypeptide of approximately 120 kDa in highly purified eIF3. Thus, adirect interaction between hPrt1 and the p170 subunit of mammalian eIF3has been demonstrated. Based on these data, the inventors conclude thathPrt1 corresponds to the second largest subunit of eIF3, p115. Theimmunoprecipitates of HA-hPrt1 did not contain other eIF3 subunits. Itis likely that HA-hPrt1 does not incorporate well into the endogenouseIF3 because of the stability of the complex. Alternatively, the HA-tagmay hinder association of HA-hPrt1 with eIF3.

Recently, Hershey and co-workers have isolated a human cDNA predicted toencode a 110 kDa protein which showed homology to the yeast Nip1protein. Although Nip1 is not present in yeast eIF3 complexes, the humanhomologue is part of mammalian eIF3. The data disclosed herein suggeststhat mammalian eIF3 contains two subunits that migrate at approximately115 kDa. Examination of various rat or rabbit eIF3 preparations resolvedon SDS-polyacrylamide gel electrophoresis show two polypeptidesmigrating at this position (Behlke, J. et al., Eur. J. Biochem.157:523-530 (1986); Meyer, L. et al., Biochemistry 21:4206-4212 (1982)).Thus, the mammalian eIF3 complex consists of at least 9 polypeptides:p170, p116 (hPrt1), p110 (hNip1), p66, p47, p44, p40, p36 and p35.

Mutations in the PRT1 gene of Saccharomyces cerevisiae impairtranslation initiation in vivo at 37° C. (Hartwell, L. and McLaughlin,C., J. Bacteriol. 96:1664-1671 (1968); Hartwell, L. and McLaughlin, C.,Proc. Natl. Acad. Sci. USA 62:468-474 (1969)). One of the mutants,prt1-1, does not promote binding of the ternary complexeIF2-GTP-tRNAimet to the 40S ribosomal subunit (Feinberg, B., et al., J.Biol. Chem. 257:10846-10851 (1982)). Evans et al. (1995) identified sixmutations in PRT1 which impair translation initiation (Evans, D. R. H.,et al., Mol. Cell. Biol. 15:4295-4535 (1995)). Two of these mutationsalter amino acids that are conserved between yeast and human Prt1. HumanPrt1, when expressed in the prt1-1 yeast strain, was unable to rescuethe temperature sensitive phenotype (N. Methot, unpublished data). Thiswas somewhat surprising since yeast eIF3 functions in a mammalianmethionyl-puromycin assay system (Naranda, T., et al., J. Biol. Chem.269:32286-32292 (1994)). Methionyl-puromycin synthesis is dependent onthe binding of the ternary complex to the 40S ribosome, requires onlywashed ribosomes, tRNAimet, eIF1A, eIF2, eIF3, eIF5 and eIF5A (Benne,R., et al., Meth. Enzymol. 60:15-35), but does not measure mRNA bindingto the ribosome. It is clear that yeast eIF3 can replace mammalian eIF3for some, but not all normal eIF3 functions, and that hPrt1 is unable tofulfill all the roles of yeast Prt1. One of the reasons why hPrt1 wasunable to replace Prt1 in vivo is that it may not incorporate into theyeast eIF3 complex. A Far Western analysis on yeast extracts using thehPrt1 N255 fragment did not reveal any interacting proteins.

Both yeast and human Prt1 contain near their amino terminus an RNArecognition motif (RRM; residues 185-270 in hPrt1, shown in FIGS. 1A-1D(SEQ ID NO:2)). The RRM contains the sequence elements that areresponsible for specific protein-protein interactions with the p170subunit of eIF3. It is unlikely that the interaction between hPrt1 andp170 is mediated through RNA since hPrt1 was unable to bind aradiolabeled RNA probe as measured by UV photocrosslinking andNorthwestern assays (N. Methot, unpublished observations). Further,treatment of the FLAG-HMK N90146 probe and the nitrocellulose membranewith RNase A did not reduce the intensity of the interaction with p170(N. Methot, unpublished data). It is possible that the RRM is functionalas an RNA binding module only within the eIF3 complex, and that its RNAbinding activity and specificity are modulated by p170. Precedents forprotein-protein interactions altering the RNA binding activity of anRRM-containing protein exist. The spliceosomal protein U2B″ is unable onits own to distinguish between the U1and U2 snRNAs, but will bindspecifically to U2 snRNA in the presence of the U2A′ protein (Scherly,D., Nature (London) 345:502-506 (1990); Scherly, D., et al., EMBO J.9:3675-3681 (1990)). U2A′ and U2B″ associate in the absence of RNA, aninteraction which is mediated by the RRM (Scherly, D., et al., EMBO J.,supra). The major RNA binding protein of yeast eIF3 is the p62 subunit(Naranda, supra). It has been previously shown that the p170 subunit ofeIF3 interacts directly with eIF4B (Methot, N., et al., Mol. Cell. Biol.in press (1996)). eIF4B and hPrt1 do not appear to interact with thesame sites on p170 since hPrt1 reacts very strongly with the 140 kDadegradation product of p170, while eIF4B does not. The numerousprotein-protein interactions involving p170 suggest that this proteinmay serve as a scaffold for both the assembly of the eIF3 complex andfor the binding of the mRNA to the ribosome.

EXAMPLE 2 Cloning of cDNAs

The cDNA #20881 was obtained from a human embryo brain cDNA library byrandom cloning. A human placenta cDNA library in γgt11 was screened witha fragment (nucleotide positions, 473 to 1200, shown in FIGS. 2A-2E (SEQID NO:3)) of cDNA #20881. 5′-RACE (rapid amplification of cDNA ends,GIBCO-BRL) was performed with HeLa poly (A)+RNA and sequence specificprimers (594 to 614 and 643 to 664 shown in FIGS. 2A-2E (SEQ ID NO:3))according to the manufacturer's instructions.

Construction of Plasmids

To generate the carboxyl (C)-terminally HA-tagged cDNAs, an antisenseprimer composed of the sequences encoding the C-terminal six amino acidsof p97 followed by the HA epitope peptide, YPYDVPDYAG, (SEQ ID NO:13)and nucleotides corresponding to the Xho I site was used for polymerasechain reaction (PCR) with a sense primer (nucleotides 2527 to 2549 shownin FIGS. 2A-2E (SEQ ID NO:3)). pcDNA3, which has a human cytomegalovirus(CMV) and T7 RNA polymerase promoters, was used as an expression vectorfor most of the experiments. pcDNA3-4-1-A(HA) and pcDNA3-6-4-A(HA)contain tile corresponding p97 cDNA sequences downstream of nucleotidepositions 12 and 30 in FIGS. 2A-2E (SEQ ID NO:3), respectively.pcDNA3-ATG-A(HA) contains the sequence downstream of nucleotide 473 anda part of the sequence of transcription factor BTEB (−15 to 10,including the initiator ATG) (Imataka, H., et al., EMBO J. 11:3633-3671(1992)).

For an N-terminally HA-tagged construct, the initiator ATG codon andthree copies of the HA sequence (Mader, S., et al., Mol. Cell. Biol.15:4990-4997 (1995)) were inserted into pcDNA3 to generate the parentalvector, pcDNA3-HA. A PCR amplified fragment from the GTG initiationcodon to a SacI site (nucleotide 600) was ligated to a fragment fromSacI to the 3′-terminus of cDNA #20881 to construct pcDNA3-HA-p97. AnEcoRI fragment of the human eIF4G cDNA (kindly provided by Dr. Rhoads;Yan, R., et al., J. Biol. Chem. 267:23226-23231 (1992)) was used toconstruct pcDNA3-HA-eIF4G. HA-La was inserted into pcDNA3 to obtainpcDNA3-HA-La.

For expression of non-tagged p97, a fragment from a BamHI restrictionsite (nucleotide 172 (FIGS. 2A-2E (SEQ ID NO:3)) to the 3′-terminus inwhich the initiator GTG had been mutated into ATG, was inserted intopcDNA3 to generate pcDNA3Bam-ATGp97. A point mutation (GTG to ATG or toGGG) was introduced using a commercial kit (Amersham). To constructpcDNA3-eIF4G, the EcoRI fragment of eIF4G cDNA was inserted into pcDNA3.

The poliovirus internal ribosome entry site (IRES) was inserted intopSP72, which contains the T7 RNA polymerase promoter, to generatepSP72IRES. For expression of p97 or eIF4G in a cap-independent manner,the Bam-ATGp97 and the EcoRI fragment of eIF4G cDNA were inserteddownstream of the IRES to construct pSP72IRES-p97 and pSP72IRES-eIF4G.

Transient Transfection

HeLa cells were infected with recombinant vaccinia virus vTF7-3 (Fuerstet al., supra), and then transfected with plasmids (5 μg) usingLipofectin (Gibco-BRL, Gaithersburg, Md.). For expression in COS-1cells, plasmids (10 μg) were transfected by electroporation (Bio-RadGene PulserII, 1200V, 25 mF).

Immunoprecipitation

After transfection, HeLa and COS-1 cells were cultured for 16 hours and48 hours, respectively, and then labeled with [³⁵S] methionine(100μCi/ml) for 1 hour in 3 cm dishes. Cells were lysed in 0.5 ml buffer A(150 mM NaCl, 1% NP-40, 0.1 % deoxycholate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM dithiothreitol (DTT), 50 mM Tris-HCl, pH 7.4).After centrifugation, the supernatant was mixed with 2 μg of anti-HAantibody (12CA5) for 6 hours in the cold room at 4° C. Protein Gsepharose (30 ml of 50% slurry) was added and the mixture was incubatedfor 2 hours. After washing with buffer A (1 ml, three times),immunoprecipitates were collected by centrifugation and proteins wereeluted with Laemmli buffer for SDS-10% polyacrylamide gelelectrophoresis (SDS-PAGE). For co-immunoprecipitation experiments,transfected HeLa cells (6 cm dish) were lysed in 1 ml buffer B (100 mMKCl, 0.5 mM EDTA, 20 mM HEPES-KOH pH 7.6, 0.4% NP-40, 20 % glycerol, 1mM DTT, 1 mM PMSF, 5 μg/ml pepstatin, 5 μg/ml leupeptin). Aftercentrifugation, an aliquot (0.5 ml) was mixed with anti-HA antibody (2μg). Immuprecipitates were washed with buffer B (1 ml, three times), andresolved by SDS-10% PAGE, except for eIF4E experiments where 12.5%polyacrylamide gels were used.

Western Blotting and Antibodies

Immunoprecipitates or cell extracts (60 μg protein) were resolved bySDS-PAGE and transferred to Immobilon polyvinylidene difluoride membrane(Millipore). Protein bands were visualized by chemiluminescence(Amersham). Quantification was done with a laser densitometer (LKB).

Anti-p97 antibodies: The peptide, SDETDSSSAPSKEQ (called INT, aminoacids 788 to 802, shown in FIGS. 2A-2E (SEQ ID NO:4)) conjugated withkeyhole limpet hemacyanin was used to raise anti-peptide(INT) antibodyin rabbits. For absorption experiments, serum was pre-incubated with theINT peptide (10 μg) on ice for 1 hour, and was used for Westernblotting. A fusion protein GST-C-terminus, glutathione-S-transferaselinked to the peptide ETAEEEESEEEAD (amino acids, 895 to 907, shown inFIGS. 2A-2E (SEQ ID NO:4)) was produced in E. coli for immunization inrabbits. The resulting serum was passed through a GST or GST-C-terminuscolumn for adsorption.

CAT Assay and RNase Protection Assay

Chloramphenicol acetyltransferase (CAT) assay was performed as described(Gorman, C. M., et al., Mol. Cell. Biol. 2:1044-1051 (1982)). RNaseprotection assay was done as described (Imataka, H., et al., EMBO J.11:3633-3671 (1992)) with modifications as follows: as an internalcontrol, in vitro synthesized unlabeled BTEB RNA and radiolabeledantisense RNA to BTEB sequence (Imataka, H., et al., EMBO J.11:3633-3671 (1992)) were mixed with antisense CAT probe. Intensities ofthe CAT and BTEB signals were quantified by Phosphorlmager BAS 2000. Theamount of CAT mRNA was normalized to that of the internal control BTEBRNA. Translation activity was calculated by dividing CAT activity by thenormalized CAT mRNA amount.

In Vitro Transcription and Translation

Capped RNA was synthesized by T7 RNA polymerase in the presence of thecap analogue, m⁷GpppG. Rabbit reticulocyte lysate (25 μl, final volume)was programmed with 0.2 μg of mRNA in the presence of [³⁵S]methionine(20 μCi) according to the manufacturer's recommendation.

Binding Assay for in Vitro Translated Factors

Following translation, five microliters of the lysate was incubated onice for 30 minutes with anti-FLAG resin (20 μl) to which FLAG-eIF4E orFLAG-eIF4A had been bound. After washing with buffer C consisting of 50mM Tris-HCl (pH, 7.5), 1 mM EDTA, 0.15 M NaCl and 0.1 % NP-40 (1 ml,three times), bound proteins were eluted with 40 μl of buffer Ccontaining 100 μg/ml FLAG peptide. pAR(DRI)[59/60] (Blanar, M. A., &Rutter, W. J., supra) was used to express FLAG-eIF4E (Pause. A., et al.,Nature 371:762-767 (1994)) and FLAG-eIF4A in E. coli.

Inducible Expression of p97

p97 cDNA (nucleotides 36 to 3810 in FIGS. 2A-2E (SEQ ID NO:3)), in whichthe initiator GTG codon was converted to ATG, was inserted into atetracycline-dependent expression vector, pRep9-CMVt (Beauparlant, P.,et al., J. Biol. Chem. 271:10690-10696 (1996)) to constructpRep9-CMVt-p97. An NIH3T3-derived cell line, S2-6 (Shockett, P., et al.,Proc. Natl. Acad. Sci. USA 92:6522-6526 (1995)) was transformed withpRep9-CMVt (control) or with pRep9-CMVt-p97 using G418 (400 μg/ml). S2-6and the established transformants were maintained in the presence of 1μg tetracycline/ml. To induce p97 expression, cells were cultured inmedium without tetracycline for 40 hours. After induction, cells wereprocessed for Western blotting or labeled with [^(2,3,5-3)H] leucine (20μCi/ml). Cells were lysed with buffer B and extracts (20 μg protein)were applied to filter paper. After washing with trichloroacetic acid(5%), radioactivity remaining on the paper was counted.

Results

The GUG-initiated Open Reading Frame Encodes a Variant of eIF4G

A human cDNA clone #20881 (hereafter called clone A, nucleotidepositions 473 to 3820 in FIGS. 2A-2E (SEQ ID NO:3)) was found to possessan open reading frame (ORF) encoding a protein (850 amino acids) similarto eIF4G. RNA synthesized from clone A produced no protein in areticulocyte lysate translation system (data not shown). The ORF ofclone A had no translation initiator ATG; the first in-frame ATG(nucleotide positions 925-927, shown in FIGS. 2A-2E (SEQ ID NO:3)) isunlikely to be the initiation codon, since there are eleven upstreamATGs which are out of frame between nucleotide positions 473 and 927.Thus, upstream sequences which could provide the translation initiatorare lacking from clone A. The 3′-terminus is complete because of thepresence of the poly (A) signal and a poly (A) tail (FIGS. 2A-2E (SEQ IDNO:3)). To obtain a full length cDNA, we screened a human placenta cDNAlibrary with a 5′ sequence of clone A. 14-1, the longest clone obtained.starts at nucleotide position 12 in FIGS. 2A-2E (SEQ ID NO:3) and thesequence was extended by 11 nucleotides by 5′-RACE. The longest sequence(3820 nucleotides) (FIGS. 2A-2E (SEQ ID NO:3)) is close to full-length,since Northern blotting showed that the mRNA is approximately 3.8 kb inlength. The mRNA is expressed in every tissue and cell line examined,implying a findamental role of the protein in cells. The first ATG ofthe extended ORF is the same as that identified in clone A (nucleotidepositions 925-927 shown in FIGS. 2A-2E (SEQ ID NO:3)) and there arein-frame stop codons at nucleotide positions 178 and 205. The sequence4-1-A (nucleotides 12 to 3820 shown in FIGS. 2A-2E (SEQ ID NO:3)) wasused for further studies.

To determine the capacity of the full length cDNA to encode a protein, amodified cDNA, 4-1-A(HA), in which the hemagglutinin (HA) epitope wasfused to the C-terminus of the ORF, was transfected into COS-1 and HeLacells. Western blotting and immunprecipitation with anti-HA antibodydemonstrated that a 97 kDa protein (called, p97-HA) was synthesized.Transfection of a truncated cDNA, ATG-A(HA), in which an artificial ATGwas inserted in frame to initiate the ORF of clone A, yielded a shorterpolypeptide than p97-HA. The apparent molecular mass of this shorterprotein is 95 kDa, which is close to the expected size from the sequenceof clone A (97 kDa, ORF of clone A plus HA). These results indicate thatthe full-length cDNA encodes a protein which is larger than that encodedby the ORF starting from position 473. One explanation for this is thattranslation of this protein starts at a non-AUG initiator 5′ upstream ofclone A. Although there is one ATG triplet in the 5′ upstream region(nucleotide positions 21-23 shown in FIGS. 2A-2E (SEQ ID NO:3)), it ispredicted to encode a small polypeptide (16 amino acids), and is out offrame of clone A ORF. Furthermore, transfection of 6-4-A(HA), which doesnot contain this ATG (FIGS. 2A-2E (SEQ ID NO:3)), produced a proteinindistinguishable from p97-HA.

We predicted that GTG at nucleotide positions 307 to 309 (FIGS. 2A-2E(SEQ ID NO:3)) is the translation initiator, since it could potentiallystart an ORF that encodes a polypeptide of about 100 kDa, and thenucleotide sequence flanking this triplet (GCCGCCAAAGUGGAG, nucleotides298-312 in FIGS. 2A-2E (SEQ ID NO:3)) is similar to the consensussequence for non-AUG initiators (Boeck, R. & Kolakofsky, D. EMBO J.13:3608-3617 (1994); Grunert, S. & Jackson, R., EMBO J. 13:3618-3630(1994)). To test this possibility, we mutated the GTG into GGG or ATG inthe 4-1-A(HA) construct, and transfected the DNA into HeLa cells. TheATG mutant yielded 4 fold more p97-HA protein than the to wild type fromsimilar amounts of RNA, while the GGG mutant failed to produce theprotein. In vitro translation experiments confirmed the in vivo results.When the 4-1-A(HA) RNA was translated in a rabbit reticulocyte lysate,p97-HA was synthesized as a single product. A point mutation (GUG toGGG) abolished translation, while a mutation to AUG increasedtranslation of p97-HA by 2 fold. From these data, we conclude thattranslation of p97 mRNA exclusively starts at the GUG codon (positions307-309 in FIGS. 2A-2E (SEQ ID NO:3)) to encode a polypeptide of 907amino acids (FIGS. 2A-2E (SEQ ID NO:3)). This mode of translation isapparently not specific to the human p97 mRNA, since the cDNA sequenceof the mouse p97 homologue also lacks an initiator ATG, and the GTGcodon is conserved.

To verify the presence of p97 protein in cells, we used two differentantisera raised against the same p97 peptide sequences as were usedabove in Western blotting. Experiments were performed with extracts froma mouse cell line Neuro2A, since anti-GST-C serum pre-incubated with GSTor GST-C detects polypeptides between 95 and 100 kDa with extracts fromprimate cell lines, including HeLa and COS-1, and these non-specificbands obscure the p97 band (data not shown). Anti-GST-C-terminal peptideantiserum detected two bands with apparent molecular masses of 97 and 65kDa, both of which disappeared when the serum was absorbed with theantigen. The 65 kDa polypeptide is likely a cross-reacting material,since another serum, anti-peptide (INT) serum, did not detect this band.In contrast the 97 kDa band was also detected by the latter serum, anddisappeared by treatment of the serum with the peptide (INT). Thus. the97 kDa polypeptide is the only common polypeptide that is specificallyrecognized by the two different antisera. To further substantiate theauthenticity of the 97 kDa polypeptide, we expressed non-tagged p97 froma cDNA. HeLa cells were employed for transfection because of theirbetter transfection efficiency. The amount of p97 was increased by 4fold following transfection with a non-tagged p97 expression plasmid,pcDNA3-Bam-ATGp97. Therefore, clearly, p97 is translated from theendogenous mRNA.

p97 Binds to eIF4A and eIF3, but not to eIF4E

Alignment of human p97 and eIF4G amino acid sequences reveals that p97exhibits 28% identity and 36% similarity to the C-terminal two thirds ofeIF4G. The N-terminal third of eIF4G, to which eIF4E binds (Lamphear,B., supra; Mader, S., et al., supra), bears no similarity to p97.Therefore, no canonical eIF4E-binding site (Mader, S., et al., supra) isfound in p97. Lamphear, B., et al., supra showed that the C-terminal twothirds of the poliovirus protease-cleaved eIF4G contains the bindingsites for eIF4A and eIF3. Thus, it is predicted that p97 would bind toeIF4A and eIF3, but not to eIF4E. To examine this, HA-tagged p97 andeIF4G were expressed in HeLa cells, and cell extracts wereimmunoprecipitated with anti-HA antibody. The immunoprecipitates wereassayed by Western blotting for eIF4A, eIF3 and HA-tagged proteinexpression. Both eIF4A and eIF3 were co-precipitated with p97 and eIF4G,while an RNA binding protein, La autoantigen (Chambers, J. C., et al.,J. Biol. Chem. 263:18043-18051 (1988)) failed to precipitate eitherfactor.

The light chain of the HA-antibody co-migrates with eIF4E on SDS-PAGE,rendering detection of immunoprecipitated eIF4E difficult. To circumventthis problem, we co-expressed FLAG-tagged eIF4E, which migrates slowerthan non-tagged eIF4E, with HA-tagged proteins. Cell extracts wereimmunoprecipitated with anti-HA, and the immunoprecipitates wereexamined by Western blotting for eIF4E and HA-tagged proteins. Nodetectable FLAG-eIF4E was co-precipitated with p97 or with La, whileeIF-4G was able to precipitate FLAG-eIF4E.

To further substantiate these results, p97 or eIF4G was synthesized invitro and mixed with bacterially expressed FLAG-eIF4E or FLAG-eIF4Abound to the anti-FLAG resin. Proteins bound to the anti-FLAG resin wereeluted with the FLAG peptide. p97 and eIF4G specifically bound to eIF4A.In contrast, binding of p97 to eIF4E was not detectable, whileinteraction between eIF4G and eIF4E was evident. La failed to bind toeither resin. We were not able to perform similar experiments for eIF3,since it consists of multi-subunits, and it is not known whichsubunit(s) interact(s) with eIF4G or p97. Thus, we conclude that p97forms a protein complex which includes eIF4A and eIF3, but excludeseIF4E.

p97 Suppresses Cap-dependent and Cap-independent Translation

Ohlmann, T., et al., EMBO J. 15:1371-1382 (1996) showed that theC-terminal two thirds of eIF4G supports cap-independent translation.Based on its homology to eIF4G, p97 might also promote cap-independenttranslation. To explore this possibility, we expressed p97 and eIF4G inHeLa cells together with a reporter CAT (chloramphenicolacetyltransferase) mRNA, whose ORF is preceded by theencephalomyocarditis virus internal ribosome entry site (EMCV-IRES).Translation of EMCV-IRES-CAT mRNA was repressed 2 fold by expression ofp97. In contrast, eIF4G stimulated cap-independent translation by 2 fold(these experiments were repeated 4 times with less than 10% variationbetween the results). Moreover, eIF4G relieved the p97-inducedrepression of translation, indicating that p97 inhibits cap-independenttranslation by competing with eIF4G. To study the effect of p97 oncap-dependent translation, CAT mRNA was used as the reporter. Similarlyto its effect on cap-independent translation, p97 inhibitedcap-dependent translation by 2 fold and the inhibition was partiallyrelieved by co-expression of eIF4G.

To study how p97 expression affects overall protein synthesis in cells,we established a cell line that expresses p97 under atetracycline-regulatable promoter (Beauparlant, P., et al., J. Biol.Chem. 271:10690-10696 (1996)). Withdrawal of tetracycline from themedium increased the amount of p97 about 4-fold without noticeablechange of the amounts of eIF4G, eIF41l or eIF4A. Overexpression of p97decreased the rate of protein synthesis by 20 to 25% as determined byincorporation of [³H] leucine. We performed similar labeling experimentswith [³⁵S] methionine and obtained essentially similar results (data notshown). These functional assays, combined with the binding results,suggest that p97 is a general suppressor of translation by forming atranslationally inactive protein complex that includes eIF4A and eIF3,but excludes eIF4E.

Discussion

The present invention further provides a new translational regulator,p97, which is homologous to the C-terminal two thirds of eIF4G. Thisregion of eIF4G contains binding sites for eIF4A and eIF3, while thebinding site for eIF4E is present in the N-terminal third of the protein(Lamphear, B., et al., supra; Mader, S., et al., supra). p97 binds toeIF4A and eIF3, but not to eIF4E. While the C-terminal two thirdsfragment of eIF4G is able to support translation initiation from theinternal ribosome entry site (IRES) of hepatitis C virus and Theiler'smurine encephalomyelitis virus (Ohlmann, T., et al., supra), p97inhibits EMCV-IRES dependent translation. It is unlikely that theopposite effects on translation are due to different IRES elements,since poliovirus IRES-mediated translation is promoted by the C-terminusof eIF4G, while transient expression of p97 repressed translation ofpoliovirus IRES-CAT mRNA. Thus, it is likely that p97 generally inhibitsIRES-dependent translation, while the C-terminal two thirds of eIF4Ggenerally supports IRES-independent translation.

p97 most likely inhibits translation by sequestration of eIF4A and eIF3,thus keeping these proteins from interacting with eIF4G. eIF4A isabsolutely required for cap-dependent and cap-independent translation(Pause, A., et al., supra) and eIF3 is essential for recruitment ofribosomes to mRNA (Pain, V. M., Eur. J. Biochem. 236:747-771 (1996)).p97 and eIF4G are likely to compete for eIF4A and eIF3 binding since,expression of eIF4G relieves p97-dependent repression of translation.This model of translational inhibition is reminiscent of the mechanismby which eIF4E-binding proteins inhibit translation. 4E-BP-1 competeswith eIF4G for binding to eIF4E, and thereby inhibits formation of thecomplete eIF4F complex (Haghighat, A., et al., EMBO J. 14:5701-5709(1995)). While 4E-BP-1 and eIF4E were reported to exist in reticulocytelysate at an approximately 1:1 molar ratio (Rau, M., et al., J. Biol.Chem. 271:8983-8990 (1996)), the present inventors could not determinethe molar ratio of p97 to other translation factors because of thedifficulty in obtaining pure recombinant protein. The relative ratios ofeIF4A, eIF4G (Duncan, R., et al., J. Biol. Chem. 262:380-388 (1987)) andeIF3 (Meyer, L. J., et al., supra; Mengod, G. & Trachsel, H. Biochem.Biophys. Acta 825:169-174 (1985)) to ribosomes in HeLa cells have beenreported to be 3, 0.2 and 0.5, respectively.

Plants have two different eIF4F complexes. One is a complex of twopolypeptides, p220 and p26, which are homologues of mammalian eIF4G andeIF4E. The other complex, called eIF(iso)4F, consists of p28, anotherhomologue of mammalian eIF4E, and p82 (Browning, K. S., et al., J. Biol.Chem. 265:17967-17973 (1990); Allen, M., et al., J. Biol. Chem.267:23232-23236 (1992)). p82 exhibits significant sequence similarity tohuman eIF4G (Allen, M., et al., supra), and the binding site for eIF4Eis present in the N-terminus of p82 (Mader, S., et al., supra).eIF(iso)4F, like eIF4F, stimulates translation in vitro (Abramson etal., J. Biol. Chem. 263:5462-5467 (1988)). Yeast also has two genesencoding eIF4G homologues, TIF4631 and TIF4632 (Goyer, C., et al., Mol.Cell. Biol. 13:4860-4874 (1993)). Although both contain an eIF4E-bindingsite (Mader, S., et al., supra), there seems to be a functionaldifference between two proteins, since TIF4631-disrupted strainsexhibited a slow-growth, cold sensitive phenotype, while disruption ofTIF4632 failed to show any phenotype. Double gene disruption engenderedlethality (Goyer, C., et al., Mol. Cell. Biol. 13:4860-4874 (1993)). Itis possible that p97 has evolved from eIF4G to become a repressor bylosing the binding site for eIF4E.

Why is GUG Employed Instead of AUG!

p97 mRNA has no initiator AUG and translation exclusively starts at aGUG codon. The nucleotide sequence surrounding the initiator GUG isGCCAAAGUGGAG (nucleotides 301-312 in FIGS. 2A-2E (SEQ ID NO:3)), whichmatches the consensus rule that purines are favorable at positions −3and +4 (the first nucleotide of the initiation codon is defined as +1,shown herein as nucleotide 307 in FIGS. 2A-2E (SEQ ID NO:3)) (Kozak, M.,J. Cell. Biol. 108:229-241 (1989)). More importantly, p97 mRNA hasadenine at the +5 position. Translation starting at a non-AUG isefficient when the second codon is GAU, where G at +4 and A at +5 aremore important than U at +6 (Boeck, R. & Kolakofsky, D. EMBO J.13:3608-3617 (1994); Grunert, S. & Jackson, R., EMBO J.13:3618-3630(1994)).

Several important regulatory genes including c-myc (Hann, S. R., et al.,Cell 52:185-195 (1988)), int-2 (Acland, P., et al., Nature 343:662-665(1990)), pim-1, FGF-2 (Florkiewicz, R. Z. & Sommer, A., Proc. Natl.Acad. Sci. USA 86:3978-3981 (1989)) and WT-1 (Bruening, W. & Pelletier,J., J. Biol. Chem. 271:8446-8454 (1996)) have non-AUG initiators inaddition to a downstream and in-frame AUG initiation codon, so thatnon-AUG initiated translation generates amino-terminally extendedproteins. Some of the extended proteins show different intracellularlocalization than their shorter counterparts (Acland, P., et al., Nature343:662-665 (1990); Bugler, B., et al., Mol. Cell. Biol. 11:573-577(1991)). In contrast, multiple products are not produced from p97 mRNA,since the GUG is the only initiator. Translation initiation at CUG ofc-Myc mRNA was enhanced, when culture medium was deprived of methionine(Hann, S. R., et al., Genes & Dev. 6:1229-1240 (1992)). For FGF-2, eIF4Fseems to activate utilization of CUG more than that of AUG (Kevil, C.,et al., Oncogene 11:2339-2348 (1996)). The expression of p97 might alsobe translationally controlled.

What is the Biological Significance of p97!

p97 is a putative modulator of interferon-γ-induced programmed celldeath. Also, apoptosis has been shown to be affected by proteinsynthesis inhibitors (Martin, D.P., et al., J. Cell Biol. 106:829-844(1988); Ledda-Columbano, G. M., et al., Am. J. Pathol. 140:545-549(1992); Polunovsky, V. A., et al., Exp. Cell Res. 214:584-594 (1994)),and overexpression of eIF4E in NIH3T3 cells prevents apoptosis inducedby serun depletion. Further, p97 mRNA is heavily edited, whenapolipoprotein B mRNA-editing protein is overexpressed in the liver oftransgenic mice, suggesting that the amount of p97 might be controlledby an editing mechanism.

EXAMPLE 3 Cloning and Expression of hPrt1 and p97 Proteins in aBaculovirus Expression System

In this illustrative example, the plasmid shuttle vector pA2 GP is usedto insert the cloned DNA encoding the hPrt1 and p97 proteins, both ofwhich lack naturally associated secretory signal (leader) sequences,into a baculovirus to express the mature proteins, using a baculovirusleader and standard methods as described in Summers et al., A Manual ofMethods for Baculovirus Vectors and Insect Cell Culture Procedures,Texas Agricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe secretory signal peptide (leader) of the baculovirus gp67 proteinand convenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpresses the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39.

The cDNA sequence encoding the hPrt1 or p97 proteins in the depositedclones, containing the AUG initiation codon is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene.

The 5′ primer for amplification of the hPrt1 coding sequence has thesequence:

5′ GACTTCTAGACCGCCATCATGCAGGACGCGGAGAACGTGGCG 3′ (SEQ ID NO:7)containing the underlined XbaI restriction enzyme site followed by 24bases of the sequence of the hPrt1 protein shown in FIGS. 1A-1D,beginning with the N-terminus of the protein. The 3′ primer has thesequence:

5′ GACTTCTAGAGGCGCAGGAGAAGGTGCCGCC 3′ (SEQ ID NO:8) containing theunderlined XbaI restriction site followed by 21 nucleotidescomplementary to the 3′ noncoding sequence shown in FIGS. 1A-1D.

The 5′ primer for amplification of the p97 coding sequence has thesequence:

5′ GACTGGTACCGCCATCATGGAGAGTGCGATTGCAGAAGGG 3′ (SEQ ID NO:9) containingthe underlined Asp718 restriction enzyme site followed by 21 bases ofthe sequence of the p97 protein shown in FIGS. 2A-2E, beginning with theN-terminus of the protein. The 3′ primer has the sequence:

5′ GACTGGTACCCGCAGTGGTTAGGTCAAATGC 3′ (SEQ ID NO:10) containing theunderlined Asp718 restriction site followed by 21 nucleotidescomplementary to the 3′ noncoding sequence shown in FIGS. 2A-2E.

The amplified fragment encoding either hPrt1 or p97 is isolated from a1% agarose gel using a commercially available kit (“Geneclean,” BIO 101Inc. La Jolla, Calif.). The hPrt1 coding fragment then is digested withXbaI and the p97 coding fragment then is digested with Asp718. Eachfragment is again is purified on a 1% agarose gel. These fragment aredesignated herein “F1”.

The plasmid is digested with the restriction enzymes with either XbaI orAsp718 and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts suchas XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with either the humanhPrt1 or p97 gene using the PCR method, in which one of the primers thatis used to amplify the gene and the second primer is from well withinthe vector so that only those bacterial colonies containing the hPrt1 orp97 gene fragment will show amplification of the DNA. The sequence ofthe cloned fragment is confirmed by DNA sequencing. These plasmids aredesignated herein pBac hPrt1 and pBac(p97).

Five μg of either pBac hPrt1 and pBac(p97) plasmid is co-transfectedwith 1.0 μg of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg ofthe plasmid are mixed in a sterile well of a microtiter plate containing50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plateb is rocked back and forthto mix the newly added solution. The plate is then incubated for 5 hoursat 27° C. After 5 hours the transfection solution is removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. The plate is put back into an incubator and cultivationis continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10). After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant viruses are called V-hPrt1 and V-p97.

To verify the expression of the hPrt1 and p97 genes, Sf9 cells are grownin Grace's medium supplemented with 10% heat inactivated FBS. The cellsare infected with the recombinant baculovirus V-hPrt1 or V-p97 at amultiplicity of infection (“MOI”) of about 2. Six hours later the mediumis removed and is replaced with SF900 II medium minus methionine andcysteine (available from Life Technologies Inc., Rockville, Md.). Ifradiolabeled proteins are desired, 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled). Microsequencing of the amino acidsequence of the amino terminus of purified protein may be used todetermine the amino terminal sequence of the mature protein and thus thecleavage point and length of the secretory signal peptide.

EXAMPLE 4 Cloning and Expression of hPrt1 and p97 in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10: 169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

EXAMPLE 4(a) Cloning and Expression in COS Cells

The expression plasmids, phPrt1 HA and p(p97) HA, are made by cloningcDNAs encoding the hPrt1 and p97 proteins into the expression vectorpcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).

The expression vector pcDNAIII contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) and a CMV promoter, apolylinker, an SV40 intron followed by a termination codon andpolyadenylation signal arranged so that a cDNA can be convenientlyplaced under expression control of the CMV promoter and operably linkedto the SV40 intron and the polyadenylation signal by means ofrestriction sites in the polylinker. The HA tag corresponds to anepitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

With respect to the hPrt1protein. a DNA fragment encoding the protein iscloned into the polylinker region of the vector so that recombinantprotein expression is directed by the CMV promoter. The plasmidconstruction strategy is as follows. The hPrt1 cDNA of the depositedclone is amplified using primers that contain convenient restrictionsites, much as described above for construction of vectors forexpression of hPrt1 protein in E. coli. Suitable primers include thefollowing, which are used in this example. The 5′ primer, containing theunderlined XbaI site, a Kozak sequence, an AUG start codon and 7additional codons of the 5′ coding region of the complete hPrt1 proteinhas the following sequence:

5′ GACTTCTAGACCGCCATCATGCAGGACGCGGAGAACGTGGCG 3′ (SEQ ID NO:7). The 3′primer, containing the underlined Xbal site, a stop codon, the HA tagsequence, and 19 bp of 3′ coding sequence has the following sequence (atthe 3′ end):

5′ GACTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTAAA TCCCCCACTGCAGACAC 3′ (SEQID NO:11).

Similarly, a DNA fragment encoding the p97 protein is cloned into thepolylinker region of the same vector. The plasmid construction strategyis as follows. The p97 cDNA of the deposited clone is also amplifiedusing primers that contain convenient restriction sites. Suitableprimers include the following, which are used in this example. The 5′primer, containing the underlined Asp718 site, a Kozak sequence, an AUGstart codon and 7 additional codons of the 5′ coding region of thecomplete p97 protein has the following sequence:

5′ GACTGGTACCGCCATCATGGAGAGTGCGATTGCAGAAGGG 3′ (SEQ ID NO:9). The 3′primer, containing the underlined Asp718 site, a stop codon, the HA tagsequence, and 18 bp of 3′ coding sequence has the following sequence (atthe 3′ end):

5′ GACGGTACCTTAAGCGTAGTCTGGGACGTCGTATGGGTAGTC AGCTTCTTCCTCTGA 3′ (SEQ IDNO:12).

The PCR amplifled DNA fragments and the vector, pcDNAIII, are digestedwith XbaI for insertion of the hPrt1 coding sequences and Asp718 forinsertion of the p97 coding sequences, and then ligated. The ligationmixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037), and the—transformed culture is plated on ampicillin mediaplates which then are incubated to allow growth of ampicillin resistantcolonies. Plasmid DNA is isolated from resistant colonies and examinedby restriction analysis or other means for the presence of either thehPrt1 or p97 encoding fragment.

For expression of recombinant hPrt1 and p97, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press (1989). Cells areincubated under conditions for expression of either hPrt1 or p97 by thevector.

Expression of the hPrt1-HA or p97-HA fusion proteins is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al. cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

EXAMPLE 4(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of hPrt1 and p97 proteins.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene, it is usually co-amplified and over-expressed. It isknown in the art that this approach may be used to develop cell linescarrying more than 1,000 copies of the amplified gene(s). Subsequently,when the methotrexate is withdrawn, cell lines are obtained whichcontain the amplified gene integrated into one or more chromosome(s) ofthe host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter is a BamHI restriction enzyme cleavage sitethat allows integration of the genes. Behind this cloning site theplasmid contains the 3′ intron and polyadenylation site of the ratpreproinsulin gene. Other high efficiency promoters can also be used forthe expression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems andsimilar systems can be used to express the hPrt1 or p97 proteins in aregulated way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc.Natl. Acad. Sci. USA 89: 5547-5551). For the polyadenylation of the mRNAother signals, e.g., from the human growth hormone or globin genes canbe used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with the restriction enzymes XbaI forinsertion of the hPrt1 encoding fragment and Asp718 for insertion of thep97 encoding fragment. The vectors are then dephosphorylated using calfintestinal phosphatase by procedures known in the art. The vectors arethen isolated from a 1% agarose gel.

The DNA sequence encoding the complete hPrt1 protein is amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe gene. The 5′ primer has the sequence:

5′ GACTTCTAGACCGCCATCATGCAGGACGCGGAGAACGTGGCG 3′ (SEQ ID NO:7)containing the underlined XbaI restriction enzyme site followed by anefficient signal for initiation of translation in eukaryotes, asdescribed by Kozak, M., J. Mol. Biol. 196:947-950 (1987), and 24 basesof the coding sequence of hPrt1 cDNA shown in FIGS. 1A-1D (SEQ ID NO:1).The 3′ primer has the sequence:

5′ GACTTCTAGAGGCGCAGGAGAAGGTGCCGCC 3 ′ (SEQ ID NO:8) containing theunderlined XbaI restriction site followed by 21 nucleotidescomplementary to the non-translated region of the hPrt1 gene shown inFIGS. 1A-1D (SEQ ID NO:1).

Similarly, the DNA sequence encoding the complete p97 protein is alsoamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the gene. The 5′ primer has the sequence:

5′ GACTGGTACCGCCATCATGGAGAGTGCGATTGCAGAAGGG 3′ (SEQ ID NO:9) containingthe underlined Asp718 restriction enzyme site followed by an efficientsignal for initiation of translation in eukaryotes, as described byKozak, M., J. Mol. Biol. 196:947-950 (1987), and 24 bases ofthe codinsequence of p97 cDNA shown in FIGS. 2A-2E (SEQ ID NO:3). The 3′ primerhas the sequence:

5′ GACTGGTACCCGCAGTGGTTAGGTCAAATGC 3′ (SEQ ID NO:10) containing theunderlined Asp718 restriction site followed by 21 nucleotidescomplementary to the non-translated region of the p97 gene shown inFIGS. 2A-2E (SEQ ID NO:3).

The amplified fragments are then digested with the endonucleases XbaIfor insertion of the hPrt1 encoding fragment and Asp718 for insertion ofthe p97 encoding fragment and then purified again on a 1% agarose gel.The isolated fragments and the dephosphorylated vectors are then ligatedwith T4 DNA ligase. E. coli HB10 or XL-1 Blue cells are then transformedand bacteria are identified that contain the fragment inserted intoplasmid pC4 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. 5 μg of the expression plasmid pC4 is cotransfected with0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al., supra).The plasmid pSV2neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

EXAMPLE 5 Tissue Distribution of hPrt1 and p97 mRNA Expression

Northern blot analysis is carried out to examine hPrt1 and p97 geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the hPrt1 (SEQ ID NO:1) or p97 (SEQ ID NO:3)protein is labeled with ³²p using the rediprime™ DNA labeling system(Amersham Life Science), according to manufacturer's instructions. Afterlabeling, the probe is purified using a CHROMA SPIN-100™ column(Clontech Laboratories, Inc.), according to manufacturer's protocolnumber PT1200-1. The purified labeled probe is then used to examinevarious human tissues for either hPrt1 or p97 mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

13 1 3032 DNA Homo sapiens CDS (97)..(2718) 1 ccctcgagtc gacggtatcgataagcttat cgataccgtc gactgctacc gaaggccggc 60 ggccgcggag ccctgcgagtaggcagcgtt gggccc atg cag gac gcg gag aac 114 Met Gln Asp Ala Glu Asn 15 gtg gcg gtg ccc gag gcg gcc gag gag cgc gcc gag ccc ggc cag cag 162Val Ala Val Pro Glu Ala Ala Glu Glu Arg Ala Glu Pro Gly Gln Gln 10 15 20cag ccg gcc gcc gag ccg ccg cca gcc gag ggg ctg ctg cgg ccc gcg 210 GlnPro Ala Ala Glu Pro Pro Pro Ala Glu Gly Leu Leu Arg Pro Ala 25 30 35 gggccc ggc gct ccg gag gcc gcg ggg acc gag gcc tcc agt gag gag 258 Gly ProGly Ala Pro Glu Ala Ala Gly Thr Glu Ala Ser Ser Glu Glu 40 45 50 gtg gggatc gcg gag gcc ggg ccg gag ccc gag gtg agg acc gag ccg 306 Val Gly IleAla Glu Ala Gly Pro Glu Pro Glu Val Arg Thr Glu Pro 55 60 65 70 gcg gccgag gca gag gcg gcc tcc ggc ccg tcc gag tcg ccc tcg ccg 354 Ala Ala GluAla Glu Ala Ala Ser Gly Pro Ser Glu Ser Pro Ser Pro 75 80 85 ccg gcc gccgag gag ctg ccc ggg tcg cat gct gag ccc cct gtc ccg 402 Pro Ala Ala GluGlu Leu Pro Gly Ser His Ala Glu Pro Pro Val Pro 90 95 100 gca cag ggcgag gcc cca gga gag cag gct cgg gac gca ggc tcc gac 450 Ala Gln Gly GluAla Pro Gly Glu Gln Ala Arg Asp Ala Gly Ser Asp 105 110 115 agc cgg gcccag gcg gtg tcc gag gac gcg gga gga aac gag ggc aga 498 Ser Arg Ala GlnAla Val Ser Glu Asp Ala Gly Gly Asn Glu Gly Arg 120 125 130 gcg gcc gaggcc gaa ccc cgg gcg ctg gag aac ggc gac gcg gac gag 546 Ala Ala Glu AlaGlu Pro Arg Ala Leu Glu Asn Gly Asp Ala Asp Glu 135 140 145 150 ccc tccttc agc gac ccc gag gac ttc gtg gac gac gtg agc gag gaa 594 Pro Ser PheSer Asp Pro Glu Asp Phe Val Asp Asp Val Ser Glu Glu 155 160 165 gaa ttactg gga gat gta ctc aaa gat cgg ccc cag gaa gca gat gga 642 Glu Leu LeuGly Asp Val Leu Lys Asp Arg Pro Gln Glu Ala Asp Gly 170 175 180 atc gattcg gtg att gta gtg gac aat gtc cct cag gtg gga ccc gac 690 Ile Asp SerVal Ile Val Val Asp Asn Val Pro Gln Val Gly Pro Asp 185 190 195 cga cttgag aaa ctc aaa aat gtc atc cac aag atc ttt tcc aag ttt 738 Arg Leu GluLys Leu Lys Asn Val Ile His Lys Ile Phe Ser Lys Phe 200 205 210 ggg aaaatc aca aat gat ttt tat cct gaa gag gat ggg aag aca aaa 786 Gly Lys IleThr Asn Asp Phe Tyr Pro Glu Glu Asp Gly Lys Thr Lys 215 220 225 230 gggtat att ttc ctg gag tac gcg tcc cct gcc cac gct gtg gat gct 834 Gly TyrIle Phe Leu Glu Tyr Ala Ser Pro Ala His Ala Val Asp Ala 235 240 245 gtgaag aac gcc gac ggc tac aag ctt gac aag cag cac aca ttc cgg 882 Val LysAsn Ala Asp Gly Tyr Lys Leu Asp Lys Gln His Thr Phe Arg 250 255 260 gtcaac ctc ttt acg gat ttt gac aag tat atg acg atc agt gac gag 930 Val AsnLeu Phe Thr Asp Phe Asp Lys Tyr Met Thr Ile Ser Asp Glu 265 270 275 tgggat att cca gag aaa cag cct ttc aaa gac ctg ggg aac tta cgt 978 Trp AspIle Pro Glu Lys Gln Pro Phe Lys Asp Leu Gly Asn Leu Arg 280 285 290 tactgg ctt gaa gag gca gaa tgc aga gat cag tac agt gtg att ttt 1026 Tyr TrpLeu Glu Glu Ala Glu Cys Arg Asp Gln Tyr Ser Val Ile Phe 295 300 305 310gag agt gga gac cgc act tcc ata ttc tgg aat gac gta aaa gac cct 1074 GluSer Gly Asp Arg Thr Ser Ile Phe Trp Asn Asp Val Lys Asp Pro 315 320 325gtc tca att gaa gaa aga gcg aga tgg aca gag acg tat gtg cgt tgg 1122 ValSer Ile Glu Glu Arg Ala Arg Trp Thr Glu Thr Tyr Val Arg Trp 330 335 340tct cct aag ggc acc tac ctg gct acc ttt cat caa aga ggc att gct 1170 SerPro Lys Gly Thr Tyr Leu Ala Thr Phe His Gln Arg Gly Ile Ala 345 350 355cta tgg ggg gga gag aaa ttc aag caa att cag aga ttc agc cac caa 1218 LeuTrp Gly Gly Glu Lys Phe Lys Gln Ile Gln Arg Phe Ser His Gln 360 365 370ggg gtt cag ctt att gac ttc tca cct tgt gaa agg tac ctg gtg acc 1266 GlyVal Gln Leu Ile Asp Phe Ser Pro Cys Glu Arg Tyr Leu Val Thr 375 380 385390 ttt agc ccc ctg atg gac acg cag gat gac cct cag gcc ata atc atc 1314Phe Ser Pro Leu Met Asp Thr Gln Asp Asp Pro Gln Ala Ile Ile Ile 395 400405 tgg gac atc ctt acg ggg cac aag aag agg ggt ttt cac tgt gag agc 1362Trp Asp Ile Leu Thr Gly His Lys Lys Arg Gly Phe His Cys Glu Ser 410 415420 tca gcc cat tgg cct att ttt aag tgg agc cat gat ggc aaa ttc ttt 1410Ser Ala His Trp Pro Ile Phe Lys Trp Ser His Asp Gly Lys Phe Phe 425 430435 gcc aga atg acc ctg gat acg ctt agc atc tat gaa act cct tct atg 1458Ala Arg Met Thr Leu Asp Thr Leu Ser Ile Tyr Glu Thr Pro Ser Met 440 445450 ggt ctt ttg gac aag aag agt ttg aag atc tct ggg ata aaa gac ttt 1506Gly Leu Leu Asp Lys Lys Ser Leu Lys Ile Ser Gly Ile Lys Asp Phe 455 460465 470 tct tgg tct cct ggt ggt aac ata atc gcc ttc tgg gtg cct gaa gac1554 Ser Trp Ser Pro Gly Gly Asn Ile Ile Ala Phe Trp Val Pro Glu Asp 475480 485 aaa gat att cca gcc agg gta acc ctg atg cag ctc cct acc agg caa1602 Lys Asp Ile Pro Ala Arg Val Thr Leu Met Gln Leu Pro Thr Arg Gln 490495 500 gag atc cga gtg agg aac ctg ttc aat gtg gtg gac tgc aag ctc cat1650 Glu Ile Arg Val Arg Asn Leu Phe Asn Val Val Asp Cys Lys Leu His 505510 515 tgg cag aag aac gga gac tac ttg tgt gtg aaa gta gat agg act ccg1698 Trp Gln Lys Asn Gly Asp Tyr Leu Cys Val Lys Val Asp Arg Thr Pro 520525 530 aaa ggc acc cag ggt gtt gtc aca aat ttt gaa att ttc cga atg agg1746 Lys Gly Thr Gln Gly Val Val Thr Asn Phe Glu Ile Phe Arg Met Arg 535540 545 550 gag aaa cag gta cct gtg gat gtg gtc gag atg aaa gaa acc atcata 1794 Glu Lys Gln Val Pro Val Asp Val Val Glu Met Lys Glu Thr Ile Ile555 560 565 gcc ttt gcc tgg gaa cca aat gga agt aag ttt gct gtg ctg cacgga 1842 Ala Phe Ala Trp Glu Pro Asn Gly Ser Lys Phe Ala Val Leu His Gly570 575 580 gag gct ccg cgg ata tct gtg tct ttc tac cac gtc aaa aac aacggg 1890 Glu Ala Pro Arg Ile Ser Val Ser Phe Tyr His Val Lys Asn Asn Gly585 590 595 aag att gaa ctc atc aag atg ttc gac aag cag cag gcg aac accatc 1938 Lys Ile Glu Leu Ile Lys Met Phe Asp Lys Gln Gln Ala Asn Thr Ile600 605 610 ttc tgg agc ccc caa gga cag ttc gtg gtg ttg gcg ggc ctg aggagt 1986 Phe Trp Ser Pro Gln Gly Gln Phe Val Val Leu Ala Gly Leu Arg Ser615 620 625 630 atg aac ggt gcc tta gcg ttt gtg gac act tcg gac tgc acggtc atg 2034 Met Asn Gly Ala Leu Ala Phe Val Asp Thr Ser Asp Cys Thr ValMet 635 640 645 aac atc gca gag cac tac atg gct tcc gac gtc gaa tgg gatcct act 2082 Asn Ile Ala Glu His Tyr Met Ala Ser Asp Val Glu Trp Asp ProThr 650 655 660 ggg cgc tac gtc gtc acc tct gtg tcc tgg tgg agc cat aaggtg gac 2130 Gly Arg Tyr Val Val Thr Ser Val Ser Trp Trp Ser His Lys ValAsp 665 670 675 aac gcg tac tgg ctg tgg act ttc cag gga cgc ctc ctg cagaag aac 2178 Asn Ala Tyr Trp Leu Trp Thr Phe Gln Gly Arg Leu Leu Gln LysAsn 680 685 690 aac aag gac cgc ttc tgc cag ctg ctg tgg cgg ccc cgg cctccc aca 2226 Asn Lys Asp Arg Phe Cys Gln Leu Leu Trp Arg Pro Arg Pro ProThr 695 700 705 710 ctc ctg agc cag gaa cag atc aag caa att aaa aag gatctg aag aaa 2274 Leu Leu Ser Gln Glu Gln Ile Lys Gln Ile Lys Lys Asp LeuLys Lys 715 720 725 tac tct aag atc ttt gaa cag aag gat cgt ttg agt cagtcc aaa gcc 2322 Tyr Ser Lys Ile Phe Glu Gln Lys Asp Arg Leu Ser Gln SerLys Ala 730 735 740 tca aag gaa ttg gtg gag aga agg cgc acc atg atg gaagat ttc cgg 2370 Ser Lys Glu Leu Val Glu Arg Arg Arg Thr Met Met Glu AspPhe Arg 745 750 755 aag tac cgg aaa atg gcc cag gag ctc tat atg gag cagaaa aac gag 2418 Lys Tyr Arg Lys Met Ala Gln Glu Leu Tyr Met Glu Gln LysAsn Glu 760 765 770 cgc ctg gag ttg cga gga ggg gtg gac act gac gag ctggac agc aac 2466 Arg Leu Glu Leu Arg Gly Gly Val Asp Thr Asp Glu Leu AspSer Asn 775 780 785 790 gtg gac gac tgg gaa gag gag acc att gag ttc ttcgtc act gaa gaa 2514 Val Asp Asp Trp Glu Glu Glu Thr Ile Glu Phe Phe ValThr Glu Glu 795 800 805 atc att ccc ctc gga atc agg agt gac ctg gag cactgt gcg cag ccg 2562 Ile Ile Pro Leu Gly Ile Arg Ser Asp Leu Glu His CysAla Gln Pro 810 815 820 tgt gtg ctg tgg agc cga ggc cgt cct gca gga agccgc gtg act ccc 2610 Cys Val Leu Trp Ser Arg Gly Arg Pro Ala Gly Ser ArgVal Thr Pro 825 830 835 gcc tcc tcc ctg tgc tct ctg gct ctg gac tgt gactgc gcc tgg att 2658 Ala Ser Ser Leu Cys Ser Leu Ala Leu Asp Cys Asp CysAla Trp Ile 840 845 850 ctg cca ttg cga cac att ttt gtg cct ttc agc ccctgg tgt ctg cag 2706 Leu Pro Leu Arg His Ile Phe Val Pro Phe Ser Pro TrpCys Leu Gln 855 860 865 870 tgg ggg att taa ggcacccgct tccacttctttcttgtttgg agttttctgt 2758 Trp Gly Ile tggaaccgcc ggcgttggct ccgaagacttagcgacgcac tggcggcacc ttctcctgcg 2818 cccagtgatg tttccacggt gcctgtacacagccgagcag catttccgtt gaaggacttg 2878 catccccatt gcgggcagtg ctggacgtgtcccggagacc caccggaggg cgccgcatgc 2938 cttgtacccc caccgtgcag gttgtggccggttttctccg caggttgaac atggaaataa 2998 aagcaaactt gtatgaaaaa aaaaaaaaaaaaaa 3032 2 873 PRT Homo sapiens 2 Met Gln Asp Ala Glu Asn Val Ala ValPro Glu Ala Ala Glu Glu Arg 1 5 10 15 Ala Glu Pro Gly Gln Gln Gln ProAla Ala Glu Pro Pro Pro Ala Glu 20 25 30 Gly Leu Leu Arg Pro Ala Gly ProGly Ala Pro Glu Ala Ala Gly Thr 35 40 45 Glu Ala Ser Ser Glu Glu Val GlyIle Ala Glu Ala Gly Pro Glu Pro 50 55 60 Glu Val Arg Thr Glu Pro Ala AlaGlu Ala Glu Ala Ala Ser Gly Pro 65 70 75 80 Ser Glu Ser Pro Ser Pro ProAla Ala Glu Glu Leu Pro Gly Ser His 85 90 95 Ala Glu Pro Pro Val Pro AlaGln Gly Glu Ala Pro Gly Glu Gln Ala 100 105 110 Arg Asp Ala Gly Ser AspSer Arg Ala Gln Ala Val Ser Glu Asp Ala 115 120 125 Gly Gly Asn Glu GlyArg Ala Ala Glu Ala Glu Pro Arg Ala Leu Glu 130 135 140 Asn Gly Asp AlaAsp Glu Pro Ser Phe Ser Asp Pro Glu Asp Phe Val 145 150 155 160 Asp AspVal Ser Glu Glu Glu Leu Leu Gly Asp Val Leu Lys Asp Arg 165 170 175 ProGln Glu Ala Asp Gly Ile Asp Ser Val Ile Val Val Asp Asn Val 180 185 190Pro Gln Val Gly Pro Asp Arg Leu Glu Lys Leu Lys Asn Val Ile His 195 200205 Lys Ile Phe Ser Lys Phe Gly Lys Ile Thr Asn Asp Phe Tyr Pro Glu 210215 220 Glu Asp Gly Lys Thr Lys Gly Tyr Ile Phe Leu Glu Tyr Ala Ser Pro225 230 235 240 Ala His Ala Val Asp Ala Val Lys Asn Ala Asp Gly Tyr LysLeu Asp 245 250 255 Lys Gln His Thr Phe Arg Val Asn Leu Phe Thr Asp PheAsp Lys Tyr 260 265 270 Met Thr Ile Ser Asp Glu Trp Asp Ile Pro Glu LysGln Pro Phe Lys 275 280 285 Asp Leu Gly Asn Leu Arg Tyr Trp Leu Glu GluAla Glu Cys Arg Asp 290 295 300 Gln Tyr Ser Val Ile Phe Glu Ser Gly AspArg Thr Ser Ile Phe Trp 305 310 315 320 Asn Asp Val Lys Asp Pro Val SerIle Glu Glu Arg Ala Arg Trp Thr 325 330 335 Glu Thr Tyr Val Arg Trp SerPro Lys Gly Thr Tyr Leu Ala Thr Phe 340 345 350 His Gln Arg Gly Ile AlaLeu Trp Gly Gly Glu Lys Phe Lys Gln Ile 355 360 365 Gln Arg Phe Ser HisGln Gly Val Gln Leu Ile Asp Phe Ser Pro Cys 370 375 380 Glu Arg Tyr LeuVal Thr Phe Ser Pro Leu Met Asp Thr Gln Asp Asp 385 390 395 400 Pro GlnAla Ile Ile Ile Trp Asp Ile Leu Thr Gly His Lys Lys Arg 405 410 415 GlyPhe His Cys Glu Ser Ser Ala His Trp Pro Ile Phe Lys Trp Ser 420 425 430His Asp Gly Lys Phe Phe Ala Arg Met Thr Leu Asp Thr Leu Ser Ile 435 440445 Tyr Glu Thr Pro Ser Met Gly Leu Leu Asp Lys Lys Ser Leu Lys Ile 450455 460 Ser Gly Ile Lys Asp Phe Ser Trp Ser Pro Gly Gly Asn Ile Ile Ala465 470 475 480 Phe Trp Val Pro Glu Asp Lys Asp Ile Pro Ala Arg Val ThrLeu Met 485 490 495 Gln Leu Pro Thr Arg Gln Glu Ile Arg Val Arg Asn LeuPhe Asn Val 500 505 510 Val Asp Cys Lys Leu His Trp Gln Lys Asn Gly AspTyr Leu Cys Val 515 520 525 Lys Val Asp Arg Thr Pro Lys Gly Thr Gln GlyVal Val Thr Asn Phe 530 535 540 Glu Ile Phe Arg Met Arg Glu Lys Gln ValPro Val Asp Val Val Glu 545 550 555 560 Met Lys Glu Thr Ile Ile Ala PheAla Trp Glu Pro Asn Gly Ser Lys 565 570 575 Phe Ala Val Leu His Gly GluAla Pro Arg Ile Ser Val Ser Phe Tyr 580 585 590 His Val Lys Asn Asn GlyLys Ile Glu Leu Ile Lys Met Phe Asp Lys 595 600 605 Gln Gln Ala Asn ThrIle Phe Trp Ser Pro Gln Gly Gln Phe Val Val 610 615 620 Leu Ala Gly LeuArg Ser Met Asn Gly Ala Leu Ala Phe Val Asp Thr 625 630 635 640 Ser AspCys Thr Val Met Asn Ile Ala Glu His Tyr Met Ala Ser Asp 645 650 655 ValGlu Trp Asp Pro Thr Gly Arg Tyr Val Val Thr Ser Val Ser Trp 660 665 670Trp Ser His Lys Val Asp Asn Ala Tyr Trp Leu Trp Thr Phe Gln Gly 675 680685 Arg Leu Leu Gln Lys Asn Asn Lys Asp Arg Phe Cys Gln Leu Leu Trp 690695 700 Arg Pro Arg Pro Pro Thr Leu Leu Ser Gln Glu Gln Ile Lys Gln Ile705 710 715 720 Lys Lys Asp Leu Lys Lys Tyr Ser Lys Ile Phe Glu Gln LysAsp Arg 725 730 735 Leu Ser Gln Ser Lys Ala Ser Lys Glu Leu Val Glu ArgArg Arg Thr 740 745 750 Met Met Glu Asp Phe Arg Lys Tyr Arg Lys Met AlaGln Glu Leu Tyr 755 760 765 Met Glu Gln Lys Asn Glu Arg Leu Glu Leu ArgGly Gly Val Asp Thr 770 775 780 Asp Glu Leu Asp Ser Asn Val Asp Asp TrpGlu Glu Glu Thr Ile Glu 785 790 795 800 Phe Phe Val Thr Glu Glu Ile IlePro Leu Gly Ile Arg Ser Asp Leu 805 810 815 Glu His Cys Ala Gln Pro CysVal Leu Trp Ser Arg Gly Arg Pro Ala 820 825 830 Gly Ser Arg Val Thr ProAla Ser Ser Leu Cys Ser Leu Ala Leu Asp 835 840 845 Cys Asp Cys Ala TrpIle Leu Pro Leu Arg His Ile Phe Val Pro Phe 850 855 860 Ser Pro Trp CysLeu Gln Trp Gly Ile 865 870 3 3820 DNA Homo sapiens CDS (307)..(3030) 3cagcagtgag tcggagctct atggaggtgg cagcgggtac cgagtggcgg ctgcagcagc 60gactcctctg agctgagttt gaggccgtcc ccgactcctt cctccccctt ccctccccct 120tttttttgtt ttccgttccc ctttcccctc ccttccctat ccccgacgac cggatcctga 180ggaggcagct gcggtggcag ctgctgagtt ctcggtgaag gtatttcatt tctcctgtcc 240cctcccctcc ccaccccatc tattaatatt attcttttga agattcttcg ttgtcaagcc 300gccaaa gtg gag agt gcg att gca gaa ggg ggt gct tct cgt ttc agt 348 ValGlu Ser Ala Ile Ala Glu Gly Gly Ala Ser Arg Phe Ser 1 5 10 gct tct tcgggc gga gga gga agt agg ggt gca cct cag cac tat ccc 396 Ala Ser Ser GlyGly Gly Gly Ser Arg Gly Ala Pro Gln His Tyr Pro 15 20 25 30 aag act gctggc aac agc gag ttc ctg ggg aaa acc cca ggg caa aac 444 Lys Thr Ala GlyAsn Ser Glu Phe Leu Gly Lys Thr Pro Gly Gln Asn 35 40 45 gct cag aaa tggatt cct gca cga agc act aga cga gat gac aac tcc 492 Ala Gln Lys Trp IlePro Ala Arg Ser Thr Arg Arg Asp Asp Asn Ser 50 55 60 gca gca aac aac tccgca aac gaa aaa gaa cga cat gat gca atc ttc 540 Ala Ala Asn Asn Ser AlaAsn Glu Lys Glu Arg His Asp Ala Ile Phe 65 70 75 agg aaa gta aga ggc atacta aat aag ctt act cct gaa aag ttt gac 588 Arg Lys Val Arg Gly Ile LeuAsn Lys Leu Thr Pro Glu Lys Phe Asp 80 85 90 aag cta tgc ctt gag ctc ctcaat gtg ggt gta gag tct aaa ctc atc 636 Lys Leu Cys Leu Glu Leu Leu AsnVal Gly Val Glu Ser Lys Leu Ile 95 100 105 110 ctt aaa ggg gtc ata ctgctg att gtg gac aaa gcc cta gaa gag cca 684 Leu Lys Gly Val Ile Leu LeuIle Val Asp Lys Ala Leu Glu Glu Pro 115 120 125 aag tat agc tca ctg tatgct cag cta tgt ctg cga ttg gca gaa gat 732 Lys Tyr Ser Ser Leu Tyr AlaGln Leu Cys Leu Arg Leu Ala Glu Asp 130 135 140 gca cca aac ttt gat ggccca gca gca gag ggt caa cca gga cag aag 780 Ala Pro Asn Phe Asp Gly ProAla Ala Glu Gly Gln Pro Gly Gln Lys 145 150 155 caa agc acc aca ttc agacgc ctc cta att tcc aaa tta caa gat gaa 828 Gln Ser Thr Thr Phe Arg ArgLeu Leu Ile Ser Lys Leu Gln Asp Glu 160 165 170 ttt gaa aac cga act agaaat gtt gat gtc tat gat aag cgt gaa aat 876 Phe Glu Asn Arg Thr Arg AsnVal Asp Val Tyr Asp Lys Arg Glu Asn 175 180 185 190 ccc ctc ctc ccc gaggag gag gaa cag aga gcc att gct aag atc aag 924 Pro Leu Leu Pro Glu GluGlu Glu Gln Arg Ala Ile Ala Lys Ile Lys 195 200 205 atg ttg gga aac atcaaa ttc att gga gag ctt ggc aag ctt gat ctt 972 Met Leu Gly Asn Ile LysPhe Ile Gly Glu Leu Gly Lys Leu Asp Leu 210 215 220 att cac gaa tct atcctt cat aag tgc atc aaa aca ctt ttg gaa aag 1020 Ile His Glu Ser Ile LeuHis Lys Cys Ile Lys Thr Leu Leu Glu Lys 225 230 235 aag aag aga gtc caactc aaa gat atg gga gag gat ttg gag tgc ctc 1068 Lys Lys Arg Val Gln LeuLys Asp Met Gly Glu Asp Leu Glu Cys Leu 240 245 250 tgt cag ata atg aggaca gtg gga cct aga tta gac cat gaa cga gcc 1116 Cys Gln Ile Met Arg ThrVal Gly Pro Arg Leu Asp His Glu Arg Ala 255 260 265 270 aag tcc tta atggat cag tac ttt gcc cga atg tgc tcc ttg atg tta 1164 Lys Ser Leu Met AspGln Tyr Phe Ala Arg Met Cys Ser Leu Met Leu 275 280 285 agt aag gaa ttgcca gca agg att cgt ttc ctg ctg cag gat acc gta 1212 Ser Lys Glu Leu ProAla Arg Ile Arg Phe Leu Leu Gln Asp Thr Val 290 295 300 gag ttg cga gaacac cat tgg gtt cct cgc aag gct ttt ctt gac aat 1260 Glu Leu Arg Glu HisHis Trp Val Pro Arg Lys Ala Phe Leu Asp Asn 305 310 315 gga cca aag acgatc aat caa att cgt caa gat gca gta aaa gat cta 1308 Gly Pro Lys Thr IleAsn Gln Ile Arg Gln Asp Ala Val Lys Asp Leu 320 325 330 ggg gtg ttt attcct gct cct atg gct caa ggg atg aga agt gac ttc 1356 Gly Val Phe Ile ProAla Pro Met Ala Gln Gly Met Arg Ser Asp Phe 335 340 345 350 ttt ctg gaggga ccg ttc atg cca ccc agg atg aaa atg gat agg gac 1404 Phe Leu Glu GlyPro Phe Met Pro Pro Arg Met Lys Met Asp Arg Asp 355 360 365 cca ctt ggagga ctt gct gat atg ttt gga caa atg cca ggt agc gga 1452 Pro Leu Gly GlyLeu Ala Asp Met Phe Gly Gln Met Pro Gly Ser Gly 370 375 380 att ggt actggt cca gga gtt atc cag gat aga ttt tca ccc acc atg 1500 Ile Gly Thr GlyPro Gly Val Ile Gln Asp Arg Phe Ser Pro Thr Met 385 390 395 gga cgt catcgt tca aat caa ctc ttc aat ggc cat ggg gga cac atc 1548 Gly Arg His ArgSer Asn Gln Leu Phe Asn Gly His Gly Gly His Ile 400 405 410 atg cct cccaca caa tcg cag ttt gga gag atg gga ggc aag ttt atg 1596 Met Pro Pro ThrGln Ser Gln Phe Gly Glu Met Gly Gly Lys Phe Met 415 420 425 430 aaa agccag ggg cta agc cag ctc tac cat aac cag agt cag gga ctc 1644 Lys Ser GlnGly Leu Ser Gln Leu Tyr His Asn Gln Ser Gln Gly Leu 435 440 445 tta tcccag ctg caa gga cag tcg aag gat atg cca cct cgg ttt tct 1692 Leu Ser GlnLeu Gln Gly Gln Ser Lys Asp Met Pro Pro Arg Phe Ser 450 455 460 aag aaagga cag ctt aat gca gat gag att agc ctg agg cct gct cag 1740 Lys Lys GlyGln Leu Asn Ala Asp Glu Ile Ser Leu Arg Pro Ala Gln 465 470 475 tcg ttccta atg aat aaa aat caa gtg cca aag ctt cag ccc cag ata 1788 Ser Phe LeuMet Asn Lys Asn Gln Val Pro Lys Leu Gln Pro Gln Ile 480 485 490 act atgatt cct cct agt gca caa cca cca cgc act caa aca cca cct 1836 Thr Met IlePro Pro Ser Ala Gln Pro Pro Arg Thr Gln Thr Pro Pro 495 500 505 510 ctggga cag aca cct cag ctt ggt ctc aaa act aat cca cca ctt atc 1884 Leu GlyGln Thr Pro Gln Leu Gly Leu Lys Thr Asn Pro Pro Leu Ile 515 520 525 caggaa aag cct gcc aag acc agc aaa aag cca cca ccg tca aag gaa 1932 Gln GluLys Pro Ala Lys Thr Ser Lys Lys Pro Pro Pro Ser Lys Glu 530 535 540 gaactc ctt aaa cta act gaa act gtt gtg act gaa tat cta aat agt 1980 Glu LeuLeu Lys Leu Thr Glu Thr Val Val Thr Glu Tyr Leu Asn Ser 545 550 555 ggaaat gca aat gag gct gtc aat ggt gta aga gaa atg agg gct cct 2028 Gly AsnAla Asn Glu Ala Val Asn Gly Val Arg Glu Met Arg Ala Pro 560 565 570 aaacac ttt ctt cct gag atg tta agc aaa gta atc atc ctg tca cta 2076 Lys HisPhe Leu Pro Glu Met Leu Ser Lys Val Ile Ile Leu Ser Leu 575 580 585 590gat aga agc gat gaa gat aaa gaa aaa gca agt tct ttg atc agt tta 2124 AspArg Ser Asp Glu Asp Lys Glu Lys Ala Ser Ser Leu Ile Ser Leu 595 600 605ctc aaa cag gaa ggg ata gcc aca agt gac aac ttc atg cag gct ttc 2172 LeuLys Gln Glu Gly Ile Ala Thr Ser Asp Asn Phe Met Gln Ala Phe 610 615 620ctg aat gta ttg gac cag tgt ccc aaa ctg gag gtt gac atc cct ttg 2220 LeuAsn Val Leu Asp Gln Cys Pro Lys Leu Glu Val Asp Ile Pro Leu 625 630 635gtg aaa tcc tat tta gca cag ttt gca gct cgt gcc atc att tca gag 2268 ValLys Ser Tyr Leu Ala Gln Phe Ala Ala Arg Ala Ile Ile Ser Glu 640 645 650ctg gtg agc att tca gaa cta gct caa cca cta gaa agt ggc acc cat 2316 LeuVal Ser Ile Ser Glu Leu Ala Gln Pro Leu Glu Ser Gly Thr His 655 660 665670 ttt cct ctc ttc cta ctt tgt ctt cag cag tta gct aaa tta caa gat 2364Phe Pro Leu Phe Leu Leu Cys Leu Gln Gln Leu Ala Lys Leu Gln Asp 675 680685 cga gaa tgg tta aca gaa ctt ttt caa caa agc aag gtc aat atg cag 2412Arg Glu Trp Leu Thr Glu Leu Phe Gln Gln Ser Lys Val Asn Met Gln 690 695700 aaa atg ctc cca gaa att gat cag aat aag gac cgc atg ttg gag att 2460Lys Met Leu Pro Glu Ile Asp Gln Asn Lys Asp Arg Met Leu Glu Ile 705 710715 ttg gaa gga aag gga ctg agt ttc tta ttc cca ctc ctc aaa ttg gag 2508Leu Glu Gly Lys Gly Leu Ser Phe Leu Phe Pro Leu Leu Lys Leu Glu 720 725730 aag gaa ctg ttg aag caa ata aag ttg gat cca tcc cct caa acc ata 2556Lys Glu Leu Leu Lys Gln Ile Lys Leu Asp Pro Ser Pro Gln Thr Ile 735 740745 750 tat aaa tgg att aaa gat aac atc tct ccc aaa ctt cat gta gat aaa2604 Tyr Lys Trp Ile Lys Asp Asn Ile Ser Pro Lys Leu His Val Asp Lys 755760 765 gga ttt gtg aac atc tta atg act agc ttc tta cag tac att tct agt2652 Gly Phe Val Asn Ile Leu Met Thr Ser Phe Leu Gln Tyr Ile Ser Ser 770775 780 gaa gta aac ccc ccc agc gat gaa aca gat tca tcc tct gct cct tcc2700 Glu Val Asn Pro Pro Ser Asp Glu Thr Asp Ser Ser Ser Ala Pro Ser 785790 795 aaa gaa cag tta gag cag gaa aaa caa cta cta cta tct ttc aag cca2748 Lys Glu Gln Leu Glu Gln Glu Lys Gln Leu Leu Leu Ser Phe Lys Pro 800805 810 gta atg cag aaa ttt ctt cat gat cac gtt gat cta caa gtc agt gcc2796 Val Met Gln Lys Phe Leu His Asp His Val Asp Leu Gln Val Ser Ala 815820 825 830 ctg tat gct ctc cag gtg cac tgc tat aac agc aac ttc cca aaaggc 2844 Leu Tyr Ala Leu Gln Val His Cys Tyr Asn Ser Asn Phe Pro Lys Gly835 840 845 atg tta ctt cgc ttt ttt gtg cac ttc tat gac atg gaa att attgaa 2892 Met Leu Leu Arg Phe Phe Val His Phe Tyr Asp Met Glu Ile Ile Glu850 855 860 gaa gaa gct ttc ttg gct tgg aaa gaa gat ata acc caa gag tttccg 2940 Glu Glu Ala Phe Leu Ala Trp Lys Glu Asp Ile Thr Gln Glu Phe Pro865 870 875 gga aaa ggc aag gct ttg ttc cag gtg aat cag tgg cta acc tggtta 2988 Gly Lys Gly Lys Ala Leu Phe Gln Val Asn Gln Trp Leu Thr Trp Leu880 885 890 gaa act gct gaa gaa gaa gaa tca gag gaa gaa gct gac taa 3030Glu Thr Ala Glu Glu Glu Glu Ser Glu Glu Glu Ala Asp 895 900 905agaaccagcc aaagccttaa attgtgcaaa acatactgtt gctatgatgt aactgcattt 3090gacctaacca ctgcgaaaat tcattccgct gtaatgtttt cacaatattt aaagcagaag 3150cacgtcagtt aggatttcct tctgcataag gtttttttgt agtgtaatgt cttaatcata 3210gtctaccatc aaatatttta ggagtatctt taatgtttag atagtatatt agcagcatgc 3270aataattaca tcataagttc tcaagcagag gcagtctatt gcaaggacct tctttgctgc 3330cagttatcat aggctgtttt aagttagaaa actgaatagc aacactgaat actgtagaaa 3390tgcactttgc tcagtaatac ttgagttgtt gcaatatttg attatccatt tggttgttac 3450agaaaaattc ttaactgtaa ttgatggttg ttgccgtaat agtatattgc ctgtatttct 3510acctctagta atgggcttta tgtgctagat tttaatatcc ttgagcctgg gcaagtgcac 3570aagtcttttt aaaagaaaca tggtttactt gcacaaaact gatcagtttt gagagatcgt 3630taatgccctt gaagtggttt ttgtgggtgt gaaacaaatg gtgagaattt gaattggtcc 3690ctcctattat agtattgaaa ttaagtctac ttaatttatc aagtcatgtt catgccctga 3750ttttatatac ttgtatctat caataaacat tgtgatactt gaaaaaaaaa aaaaaaaaaa 3810aaaaaaaaaa 3820 4 907 PRT Homo sapiens 4 Val Glu Ser Ala Ile Ala Glu GlyGly Ala Ser Arg Phe Ser Ala Ser 1 5 10 15 Ser Gly Gly Gly Gly Ser ArgGly Ala Pro Gln His Tyr Pro Lys Thr 20 25 30 Ala Gly Asn Ser Glu Phe LeuGly Lys Thr Pro Gly Gln Asn Ala Gln 35 40 45 Lys Trp Ile Pro Ala Arg SerThr Arg Arg Asp Asp Asn Ser Ala Ala 50 55 60 Asn Asn Ser Ala Asn Glu LysGlu Arg His Asp Ala Ile Phe Arg Lys 65 70 75 80 Val Arg Gly Ile Leu AsnLys Leu Thr Pro Glu Lys Phe Asp Lys Leu 85 90 95 Cys Leu Glu Leu Leu AsnVal Gly Val Glu Ser Lys Leu Ile Leu Lys 100 105 110 Gly Val Ile Leu LeuIle Val Asp Lys Ala Leu Glu Glu Pro Lys Tyr 115 120 125 Ser Ser Leu TyrAla Gln Leu Cys Leu Arg Leu Ala Glu Asp Ala Pro 130 135 140 Asn Phe AspGly Pro Ala Ala Glu Gly Gln Pro Gly Gln Lys Gln Ser 145 150 155 160 ThrThr Phe Arg Arg Leu Leu Ile Ser Lys Leu Gln Asp Glu Phe Glu 165 170 175Asn Arg Thr Arg Asn Val Asp Val Tyr Asp Lys Arg Glu Asn Pro Leu 180 185190 Leu Pro Glu Glu Glu Glu Gln Arg Ala Ile Ala Lys Ile Lys Met Leu 195200 205 Gly Asn Ile Lys Phe Ile Gly Glu Leu Gly Lys Leu Asp Leu Ile His210 215 220 Glu Ser Ile Leu His Lys Cys Ile Lys Thr Leu Leu Glu Lys LysLys 225 230 235 240 Arg Val Gln Leu Lys Asp Met Gly Glu Asp Leu Glu CysLeu Cys Gln 245 250 255 Ile Met Arg Thr Val Gly Pro Arg Leu Asp His GluArg Ala Lys Ser 260 265 270 Leu Met Asp Gln Tyr Phe Ala Arg Met Cys SerLeu Met Leu Ser Lys 275 280 285 Glu Leu Pro Ala Arg Ile Arg Phe Leu LeuGln Asp Thr Val Glu Leu 290 295 300 Arg Glu His His Trp Val Pro Arg LysAla Phe Leu Asp Asn Gly Pro 305 310 315 320 Lys Thr Ile Asn Gln Ile ArgGln Asp Ala Val Lys Asp Leu Gly Val 325 330 335 Phe Ile Pro Ala Pro MetAla Gln Gly Met Arg Ser Asp Phe Phe Leu 340 345 350 Glu Gly Pro Phe MetPro Pro Arg Met Lys Met Asp Arg Asp Pro Leu 355 360 365 Gly Gly Leu AlaAsp Met Phe Gly Gln Met Pro Gly Ser Gly Ile Gly 370 375 380 Thr Gly ProGly Val Ile Gln Asp Arg Phe Ser Pro Thr Met Gly Arg 385 390 395 400 HisArg Ser Asn Gln Leu Phe Asn Gly His Gly Gly His Ile Met Pro 405 410 415Pro Thr Gln Ser Gln Phe Gly Glu Met Gly Gly Lys Phe Met Lys Ser 420 425430 Gln Gly Leu Ser Gln Leu Tyr His Asn Gln Ser Gln Gly Leu Leu Ser 435440 445 Gln Leu Gln Gly Gln Ser Lys Asp Met Pro Pro Arg Phe Ser Lys Lys450 455 460 Gly Gln Leu Asn Ala Asp Glu Ile Ser Leu Arg Pro Ala Gln SerPhe 465 470 475 480 Leu Met Asn Lys Asn Gln Val Pro Lys Leu Gln Pro GlnIle Thr Met 485 490 495 Ile Pro Pro Ser Ala Gln Pro Pro Arg Thr Gln ThrPro Pro Leu Gly 500 505 510 Gln Thr Pro Gln Leu Gly Leu Lys Thr Asn ProPro Leu Ile Gln Glu 515 520 525 Lys Pro Ala Lys Thr Ser Lys Lys Pro ProPro Ser Lys Glu Glu Leu 530 535 540 Leu Lys Leu Thr Glu Thr Val Val ThrGlu Tyr Leu Asn Ser Gly Asn 545 550 555 560 Ala Asn Glu Ala Val Asn GlyVal Arg Glu Met Arg Ala Pro Lys His 565 570 575 Phe Leu Pro Glu Met LeuSer Lys Val Ile Ile Leu Ser Leu Asp Arg 580 585 590 Ser Asp Glu Asp LysGlu Lys Ala Ser Ser Leu Ile Ser Leu Leu Lys 595 600 605 Gln Glu Gly IleAla Thr Ser Asp Asn Phe Met Gln Ala Phe Leu Asn 610 615 620 Val Leu AspGln Cys Pro Lys Leu Glu Val Asp Ile Pro Leu Val Lys 625 630 635 640 SerTyr Leu Ala Gln Phe Ala Ala Arg Ala Ile Ile Ser Glu Leu Val 645 650 655Ser Ile Ser Glu Leu Ala Gln Pro Leu Glu Ser Gly Thr His Phe Pro 660 665670 Leu Phe Leu Leu Cys Leu Gln Gln Leu Ala Lys Leu Gln Asp Arg Glu 675680 685 Trp Leu Thr Glu Leu Phe Gln Gln Ser Lys Val Asn Met Gln Lys Met690 695 700 Leu Pro Glu Ile Asp Gln Asn Lys Asp Arg Met Leu Glu Ile LeuGlu 705 710 715 720 Gly Lys Gly Leu Ser Phe Leu Phe Pro Leu Leu Lys LeuGlu Lys Glu 725 730 735 Leu Leu Lys Gln Ile Lys Leu Asp Pro Ser Pro GlnThr Ile Tyr Lys 740 745 750 Trp Ile Lys Asp Asn Ile Ser Pro Lys Leu HisVal Asp Lys Gly Phe 755 760 765 Val Asn Ile Leu Met Thr Ser Phe Leu GlnTyr Ile Ser Ser Glu Val 770 775 780 Asn Pro Pro Ser Asp Glu Thr Asp SerSer Ser Ala Pro Ser Lys Glu 785 790 795 800 Gln Leu Glu Gln Glu Lys GlnLeu Leu Leu Ser Phe Lys Pro Val Met 805 810 815 Gln Lys Phe Leu His AspHis Val Asp Leu Gln Val Ser Ala Leu Tyr 820 825 830 Ala Leu Gln Val HisCys Tyr Asn Ser Asn Phe Pro Lys Gly Met Leu 835 840 845 Leu Arg Phe PheVal His Phe Tyr Asp Met Glu Ile Ile Glu Glu Glu 850 855 860 Ala Phe LeuAla Trp Lys Glu Asp Ile Thr Gln Glu Phe Pro Gly Lys 865 870 875 880 GlyLys Ala Leu Phe Gln Val Asn Gln Trp Leu Thr Trp Leu Glu Thr 885 890 895Ala Glu Glu Glu Glu Ser Glu Glu Glu Ala Asp 900 905 5 33 DNA Homosapiens 5 accggaattc aaaatggacg cggacgagcc ctc 33 6 28 DNA Homo sapiens6 agcggaattc ttaaatcccc cactgcag 28 7 42 DNA Homo sapiens 7 gacttctagaccgccatcat gcaggacgcg gagaacgtgg cg 42 8 31 DNA Homo sapiens 8gacttctaga ggcgcaggag aaggtgccgc c 31 9 40 DNA Homo sapiens 9 gactggtaccgccatcatgg agagtgcgat tgcagaaggg 40 10 31 DNA Homo sapiens 10 gactggtacccgcagtggtt aggtcaaatg c 31 11 58 DNA Homo sapiens 11 gactctagattaagcgtagt ctgggacgtc gtatgggtaa atcccccact gcagacac 58 12 57 DNA Homosapiens 12 gacggtacct taagcgtagt ctgggacgtc gtatgggtag tcagcttcttcctctga 57 13 10 PRT Homo sapiens 13 Tyr Pro Tyr Asp Val Pro Asp Tyr AlaGly 1 5 10

What is claimed is:
 1. An isolated polynucleotide comprising a nucleicacid encoding an amino acid sequence at least 90% identical to aminoacids 2 to 873 of SEQ ID NO:2.
 2. The polynucleotide of claim 1,comprising anucleic acid encoding an amino acid sequence at least 95%identical to amino acids 2 to 873 of SEQ ID NO:2.
 3. The polynucleotideof claim 2, comprising a nucleic acid encoding amino acids 2 to 873 ofSEQ ID NO:2.
 4. The polynucleotide of claim 3, comprising nucleotides100 to 2715 of SEQ ID NO:1.
 5. The polynucleotide of claim 1, comprisinga nucleic acid encoding an amino acid sequence at least 90% identical toamino acids 1 to 873 of SEQ ID NO:2.
 6. The polynucleotide of claim 5,comprising a nucleic acid encoding an amino acid sequence at least 95%identical to amino acids 1 to 873 of SEQ ID NO:2.
 7. The polynucleotideof claim 6, comprising a nucleic acid encoding amino acids 1 to 873 ofSEQ ID NO:2.
 8. The polynucleotide of claim 7, comprising nucleotides 97to 2715 of SEQ ID NO:1.
 9. The polynucleotide of claim 1, comprising anucleic acid which encodes a protein having a biological activityselected from the group consisting of: (i) binding affinity for the p170subunit of eIF3; and (ii) binding activity for an antibody havingspecificity for a protein consisting of the complete amino acid sequenceof SEQ ID NO:2.
 10. The polynucleotide of claim 1, further comprising aheterologous polynucleotide.
 11. The polynucleotide of claim 10, whereinthe heterologous polynucleotide encodes a heterologous protein.
 12. Amethod of producing a vector which comprises inserting thepolynucleotide of claim 1 into a vector.
 13. A vector comprising thepolynucleotide of claim
 1. 14. The vector of claim 13, wherein thepolynucleotide is operably linked to a heterologous regulatorypolynucleotide.
 15. A host cell comprising the polynucleotide of claim1.
 16. The host cell of claim 15, wherein the polynucleotide is operablylinked to a heterologous regulatory polynucleotide.
 17. A method ofproducing a protein comprising an amino acid sequence at least 90%identical to amino acids 2 to 873 of SEQ ID NO:2 comprising culturingthe host cell of claim 16 under conditions such that the protein isexpressed, and recovering the protein.
 18. An isolated polynucleotidecomprising a nucleic acid encoding an amino acid sequence at least 90%identical to the amino acid sequence encoded by the cDNA in ATCC DepositNo.
 97766. 19. The polynucleotide of claim 18, comprising anucleic acidencoding an amino acid sequence at least 95% identical to the amino acidsequence encoded by the cDNA in ATCC Deposit No.
 97766. 20. Thepolynucleotide of claim 19, comprising a nucleic acid encoding the aminoacid sequence encoded by the cDNA in ATCC Deposit No.
 97766. 21. Thepolynucleotide of claim 18, comprising a nucleic acid which encodes aprotein having a biological activity selected from the group consistingof: (i) binding affinity for the p170 subunit of eIF3; and (ii) bindingactivity for an antibody having specificity for a protein consisting ofthe amino acid sequence encoded by the cDNA in ATCC Deposit No. 97766.22. The polynucleotide of claim 18, further comprising a heterologouspolynucleotide.
 23. The polynucleotide of claim 22, wherein theheterologous polynucleotide encodes a heterologous protein.
 24. A methodof producing a vector which comprises inserting the polynucleotide ofclaim 18 into a vector.
 25. A vector comprising the polynucleotide ofclaim
 18. 26. The vector of claim 25, wherein the polynucleotide isoperably linked to a heterologous regulatory polynucleotide.
 27. A hostcell comprising the polynucleotide of claim
 18. 28. The host cell ofclaim 27, wherein the polynucleotide is operably linked to aheterologous regulatory polynucleotide.
 29. A method of producing aprotein comprising an amino acid sequence at least 90% identical to theamino acid sequence encoded by the cDNA in ATCC Deposit No. 97766comprising culturing the host cell of claim 28 under conditions suchthat the protein is expressed, and recovering the protein.
 30. Anisolated polynucleotide comprising a nucleic acid which encodes a firstamino acid sequence at least 90% identical to a second amino acidsequence selected from the group consisting of: (a) amino acids 1 to 188of SEQ ID NO:2; (b) amino acids 193 to 235 of SEQ ID NO:2; (c) aminoacids 248 to 262 of SEQ ID NO:2; and (d) amino acids 270 to 350 of SEQID NO:2.
 31. The polynucleotide of claim 30, comprising a nucleic acidwhich encodes a first amino acid sequence at least 90% identical toamino acids 1 to 188 of SEQ ID NO:2.
 32. The polynucleotide of claim 31,comprising a nucleic acid which encodes a first amino acid sequence atleast 95% identical to amino acids 1 to 188 of SEQ ID NO:2.
 33. Thepolynucleotide of claim 32, comprising a nucleic acid which encodes afirst amino acid sequence identical to amino acids 1 to 188 of SEQ IDNO:2.
 34. The polynucleotide of claim 33, comprising nucleotides 97 to660 of SEQ ID NO:1.
 35. The polynucleotide of claim 30, comprising anucleic acid which encodes a first amino acid sequence at least 90%identical to amino acids 193 to 235 of SEQ ID NO:2.
 36. Thepolynucleotide of claim 35, comprising a nucleic acid which encodes afirst amino acid sequence at least 95% identical to amino acids 193 to235 of SEQ ID NO:2.
 37. The polynucleotide of claim 36, comprising anucleic acid which encodes a first amino acid sequence identical toamino acids 193 to 235 of SEQ ID NO:2.
 38. The polynucleotide of claim37, comprising nucleotides 673 to 801 of SEQ ID NO:1.
 39. Thepolynucleotide of claim 30, comprising a nucleic acid which encodes afirst amino acid sequence at least 90% identical to amino acids 248 to262 of SEQ ID NO:2.
 40. The polynucleotide of claim 39, comprising anucleic acid which encodes a first amino acid sequence at least 95%identical to amino acids 248 to 262 of SEQ ID NO:2.
 41. Thepolynucleotide of claim 40, comprising a nucleic acid which encodes afirst amino acid sequence identical to amino acids 248 to 262 of SEQ IDNO:2.
 42. The polynucleotide of claim 41, comprising nucleotides 838 to882 of SEQ ID NO:1.
 43. The polynucleotide of claim 30, comprising anucleic acid which encodes a first amino acid sequence at least 90%identical to amino acids 270 to 350 of SEQ ID NO:2.
 44. Thepolynucleotide of claim 43, comprising a nucleic acid which encodes afirst amino acid sequence at least 95% identical to amino acids 270 to350 of SEQ ID NO:2.
 45. The polynucleotide of claim 44, comprising anucleic acid which encodes a first amino acid sequence identical toamino acids 270 to 350 of SEQ ID NO:2.
 46. The polynucleotide of claim45, comprising nucleotides 904 to 1146 of SEQ ID NO:1.
 47. Thepolynucleotide of claim 30, comprising a nucleic acid which encodes afirst amino acid sequence identical to amino acids 248 to 262 of SEQ IDNO:2 and amino acids 270 to 350 of SEQ ID NO:2.
 48. The polynucleotideof claim 30, comprising a nucleic acid which encodes a protein having abiological activity selected from the group consisting of: (i) bindingaffinity for the p170 subunit of eIF3; and (ii) binding activity for anantibody having specificity for a protein consisting of the completeamino acid sequence of SEQ ID NO:2.
 49. The polynucleotide of claim 30,further comprising aheterologous polynucleotide.
 50. The polynucleotideof claim 49, wherein the heterologous polynucleotide encodes aheterologous protein.
 51. A method of producing a vector which comprisesinserting the polynucleotide of claim 30 into a vector.
 52. A vectorcomprising the polynucleotide of claim
 30. 53. The vector of claim 52,wherein the polynucleotide is operably linked to a heterologousregulatory polynucleotide.
 54. A host cell comprising the polynucleotideof claim
 30. 55. The host cell of claim 54, wherein the polynucleotideis operably linked to a heterologous regulatory polynucleotide.
 56. Amethod ofproducing a protein comprising culturing the host cell of claim55 under conditions such that the protein is expressed, and recoveringthe protein, wherein said protein has a first amino acid sequence atleast 90% identical to a second amino acid sequence selected from thegroup consisting of: (a) amino acids 1 to 188 of SEQ ID NO:2; (b) aminoacids 193 to 235 of SEQ ID NO:2; (c) amino acids 248 to 262 of SEQ IDNO:2; and (d) amino acids 270 to 350 of SEQ ID NO:2.
 57. An isolatedpolynucleotide comprising a nucleic acid which encodes a first aminoacid sequence at least 90% identical to a second amino acid sequenceselected from the group consisting of: (a) amino acids 361 to 449 of SEQID NO:2; (b) amino acids 458 to 620 of SEQ ID NO:2; and (c) amino acids639 to 846 of SEQ ID NO:2.
 58. The polynucleotide of claim 57,comprising a nucleic acid which encodes a first amino acid sequence atleast 90% identical to amino acids 361 to 449 of SEQ ID NO:2.
 59. Thepolynucleotide of claim 58, comprising a nucleic acid which encodes afirst amino acid sequence at least 95% identical to amino acids 361 to449 of SEQ ID NO:2.
 60. The polynucleotide of claim 59, comprising anucleic acid which encodes a first amino acid sequence identical toamino acids 361 to 449 of SEQ ID NO:2.
 61. The polynucleotide of claim60, comprising nucleotides 1177 to 1443 of SEQ ID NO:1.
 62. Thepolynucleotide of claim 57, comprising a nucleic acid which encodes afirst amino acid sequence at least 90% identical to amino acids 458 to620 of SEQ ID NO:2.
 63. The polynucleotide of claim 62, comprising anucleic acid which encodes a first amino acid sequence at least 95%identical to amino acids 458 to 620 of SEQ ID NO:2.
 64. Thepolynucleotide of claim 63, comprising a nucleic acid which encodes afirst amino acid sequence identical to amino acids 458 to 620 of SEQ IDNO:2.
 65. The polynucleotide of claim 64, comprising nucleotides 1468 to1956 of SEQ ID NO:1.
 66. The polynucleotide of claim 57, comprising anucleic acid which encodes a first amino acid sequence at least 90%identical to amino acids 639 to 846 of SEQ ID NO:2.
 67. Thepolynucleotide of claim 66, comprising a nucleic acid which encodes afirst amino acid sequence at least 95% identical to amino acids 639 to846 of SEQ ID NO:2.
 68. The polynucleotide of claim 67, comprising anucleic acid which encodes a first amino acid sequence identical toamino acids 639 to 846 of SEQ ID NO:2.
 69. The polynucleotide of claim68, comprising nucleotides 2011 to 2634 of SEQ ID NO:1.
 70. Thepolynucleotide of claim 57, comprising a nucleic acid which encodes afirst amino acid sequence identical to amino acids 361 to 449 of SEQ IDNO:2 and amino acids 458 to 620 of SEQ ID NO:2.
 71. The polynucleotideof claim 57, comprising a nucleic acid which encodes a protein having abiological activity selected from the group consisting of: (i) bindingaffinity for the p170 subunit of eIF3; and (ii) binding activity for anantibody having specificity for a protein consisting of the completeamino acid sequence of SEQ ID NO:2.
 72. The polynucleotide of claim 57,further comprising a heterologous polynucleotide.
 73. The polynucleotideof claim 72, wherein the heterologous polynucleotide encodes aheterologous protein.
 74. A method of producing a vector which comprisesinserting the polynucleotide of claim 57 into a vector.
 75. A vectorcomprising the polynucleotide of claim
 57. 76. The vector of claim 75,wherein the polynucleotide is operably linked to a heterologousregulatory polynucleotide.
 77. A host cell comprising the polynucleotideof claim
 57. 78. The host cell of claim 77, wherein the polynucleotideis operably linked to a heterologous regulatory polynucleotide.
 79. Amethod ofproducing aprotein comprising culturing the host cell of claim78 under conditions such that the protein is expressed, and recoveringthe protein, wherein said protein has a first amino acid sequence atleast 90% identical to a second amino acid sequence selected from thegroup consisting of: (a) amino acids 361 to 449 of SEQ ID NO:2; (b)amino acids 458 to 620 of SEQ ID NO:2; and (c) amino acids 639 to 846 ofSEQ ID NO:2.
 80. An isolated polynucleotide comprising a nucleic acidwhich encodes a first amino acid sequence at least 90% identical to asecond amino acid sequence selected from the group consisting of: (a)amino acids 147 to 255 in SEQ ID NO:2; and (b) amino acids 185 to 270 inSEQ ID NO:2.
 81. The polynucleotide of claim 80, comprising a nucleicacid which encodes a first amino acid sequence at least 90% identical toamino acids 147 to 255 in SEQ ID NO:2.
 82. The polynucleotide of claim81, comprising a nucleic acid which encodes a first amino acid sequenceat least 95% identical to amino acids 147 to 255 in SEQ ID NO:2.
 83. Thepolynucleotide of claim 82, comprising a nucleic acid which encodes afirst amino acid sequence identical to amino acids 147 to 255 in SEQ IDNO:2.
 84. The polynucleotide of claim 83, comprising nucleotides 535 to711 of SEQ ID NO:1.
 85. The polynucleotide of claim 80, comprising anucleic acid which encodes a first amino acid sequence at least 90%identical to amino acids 185 to 270 in SEQ ID NO:2.
 86. Thepolynucleotide of claim 85, comprising a nucleic acid which encodes afirst amino acid sequence at least 95% identical to amino acids 185 to270 in SEQ ID NO:2.
 87. The polynucleotide of claim 86, comprising anucleic acid which encodes a first amino acid sequence identical toamino acids 185 to 270 in SEQ ID NO:2.
 88. The polynucleotide of claim87, comprising nucleotides 649 to 906 of SEQ ID NO:1.
 89. Thepolynucleotide of claim 80, comprising a nucleic acid which encodes aprotein having a biological activity selected from the group consistingof: (i) binding affinity for the p170 subunit of eIF3; and (ii) bindingactivity for an antibody having specificity for a protein consisting ofthe complete amino acid sequence of SEQ ID NO:2.
 90. The polynucleotideof claim 80, further comprising a heterologous polynucleotide.
 91. Thepolynucleotide of claim 90, wherein the heterologous polynucleotideencodes a heterologous protein.
 92. A method of producing a vector whichcomprises inserting the polynucleotide of claim 80 into a vector.
 93. Avector comprising the polynucleotide of claim
 80. 94. The vector ofclaim 93, wherein the polynucleotide is operably linked to aheterologous regulatory polynucleotide.
 95. A host cell comprising thepolynucleotide of claim
 80. 96. The host cell of claim 95, wherein thepolynucleotide is operably linked to a heterologous regulatorypolynucleotide.
 97. A method ofproducing a protein comprising culturingthe host cell of claim 96 under conditions such that the protein isexpressed, and recovering the protein, wherein said protein has a firstamino acid sequence at least 90% identical to a second amino acidsequence selected from the group consisting of: (a) amino acids 147 to255 in SEQ ID NO:2; and (b) amino acids 185 to 270 in SEQ ID NO:2.